Process for displacing an exogenous enzyme

ABSTRACT

The present disclosure concerns a process for fermenting a biomass with a reduced dose of a purified exogenous enzyme (which can be, for example a purified exogenous glucoamylase). The process comprises contacting a biomass (which may comprise starch) with a recombinant yeast host cell. The recombinant yeast host cell has a genetic modification for expressing a heterologous polypeptide having starch or dextrin hydrolase activity (which may be, for example, from a glucoamylase). The nucleic acid molecule encoding the heterologous polypeptide having starch or dextrin hydrolase activity comprises allowing the secretion of the heterologous polypeptide.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 580127_42601_SEQUENCE_LISTING.txt. The text fileis 155 KB, was created on Nov. 25, 2020, and is being submittedelectronically via EFS-Web.

TECHNOLOGICAL FIELD

The present disclosure relates to process for displacing, at least inpart, an exogenous enzyme during a fermentation using a recombinantyeast host cell expressing a heterologous starch or dextrin hydrolase.

BACKGROUND

Saccharomyces cerevisiae is the primary biocatalyst used in thecommercial production of fuel ethanol. This organism is proficient infermenting glucose to ethanol, often to concentrations greater than 20%v/v. However, S. cerevisiae lacks the ability to hydrolyzepolysaccharides. Consequently, in addition to yeast, industrial ethanolproduction requires the exogenous addition of expensive enzymes toconvert complex sugars to glucose. For example, in the United States,the primary source of fuel ethanol is corn starch. Regardless of themashing process, corn starch fermentation by yeast benefits from theexogenous addition of both α-amylase and glucoamylase.

The fermentation processes employed in the corn ethanol industry can bebroadly classified based on utilized substrate into liquefied corn mashand raw corn flour fermentations. In the mashing process, corn is boththermally and enzymatically liquefied prior to fermentation usingα-amylase, which breaks down long chain starch polymers into smallerdextrins. The mash is then cooled and inoculated with S. cerevisiae.Concomitantly, the exogenous purified glucoamylase is added.Glucoamylases (GAs) break down the branched dextrin into glucosemolecules that are utilizable by yeast. GAs primarily hydrolyzeα-1,4-glycosidic linkages from non-reducing ends in starch chain (theyare, hence, exo-acting enzymes), while α-amylases can also hydrolyzeα-1,6-glicosidic linkages from the inner starch chains (and are,therefore, endo-acting enzymes). The availability of a robust,ethanol-tolerant yeast strain is required to ferment the hydrolyzedstarch into the desired final product, ethanol.

In dry mill fuel ethanol production from grain, incoming starch-richgrain (typically corn, but also milo or wheat) can be reduced in size ina hammer or roller mill to create a flour, which can be blended withwater, recycled thin stillage (“backset”), and exogenous alpha amylaseenzymes to create a slurry. This slurry can be heated with steam andheld at elevated temperature during a mashing or cooking process togelatinize the starch and allow hydrolysis of the starch by thealpha-amylase enzymes to convert the starch to a range of soluble andinsoluble dextrins. Having gone through this cooking process, the slurry(now referred to as “liquefact” or “mash”) can be cooled to fermentationtemperatures and used to fill fermentors for the production of fuelethanol by yeast fermentation.

Yeast (especially Saccharomyces cerevisiae) can be added to fermentationafter having been allowed to grow on a portion of the liquefact in aseparate vessel for several generations in order to increase the totalnumber of yeast cells added to fermentation and to acclimate the yeastto the liquefact in a process referred to as “mash propagation”;however, yeasts can also be added directly to fermentation without thisadditional growth step in a process variation known as “direct pitch.”Nitrogen for growth of the yeast can be provided to fermentation throughamino acids in the liquefact (the concentration of which is sometimesincreased through addition of exogenous protease enzymes infermentation), the addition of urea to fermentation, and/or the additionof ammonia directly to fermentation or to the mash during the cookingprocess. The state of the art is that exogenous glucoamylase (GA) enzymecocktails can be added to fermentation to hydrolyze the starch dextrinsin the liquefact to glucose, allowing for uptake and fermentation of theglucose to ethanol by the yeast. This process is typically referred toas simultaneous saccharification and fermentation (SSF). Combination ofthese components in fermentation results in a fermenting slurry referredto as “beer.” Exogenous GA enzyme cocktails are purchased for thispurpose by fuel ethanol producers at substantial cost, generally asconcentrated products in a stabilized formulation having undergone somelevel of filtration or purification from the enzyme production culture.For production facilities using mash propagation to increase the totalnumber of yeast cells added to fermentation, a small amount of exogenousGA enzyme cocktail can be added to the mash in propagation in order torelease glucose to provide for growth of the yeast cells; the volume ofexogenous GA enzyme cocktail added to each mash propagation batch istypically less than 3 gallons, or typically less than 2.5×10⁻⁴ v/v(volume of exogenous GA enzyme cocktail per total volume of mashpropagation batch).

It would be highly desirable to be provided with improved and/or moreefficient yeast strains which reduces or precludes the need for highlyexpensive enzyme purification and formulation, thus significantlyreducing overall production costs.

BRIEF SUMMARY

The present disclosure provides recombinant yeast host cells whichexpresses starch digesting glucoamylases that can be used insaccharification and fermentation of a biomass. The present disclosureconcerns recombinant yeast host cells expressing a heterologous starchdigesting glucoamylase by introducing a heterologous nucleic acidmolecule encoding for the glucoamylase enzyme as well as a signalsequence allowing the secretion of the glucoamylase. The presentdisclosure also provides a process for making a fermentation productfrom a biomass while reducing the dose of an exogenous enzyme tocomplete the fermentation.

According to a first aspect, the present disclosure provides arecombinant yeast host cell for saccharification and fermentation of abiomass, the recombinant yeast host cell having a heterologous nucleicacid molecule encoding a heterologous polypeptide having glucoamylaseactivity. The heterologous nucleic acid molecule comprises a firstpolynucleotide encoding a heterologous signal sequence wherein theheterologous signal sequence has the amino acid sequence of SEQ ID NO:5, is a variant of the amino acid sequence of SEQ ID NO: 5 having signalsequence activity, or is a fragment of the amino acid sequence of SEQ IDNO: 5 having signal sequence activity. The heterologous nucleic acidalso comprises a second polynucleotide encoding the heterologouspolypeptide having glucoamylase activity, wherein the polypeptide havingglucoamylase activity has the amino acid sequence of SEQ ID NO: 3, 13 or27 to 36, is a variant of the amino acid sequence of SEQ ID NO: 3, 13 or27 to 36 having glucoamylase activity, or is a fragment of the aminoacid sequence of SEQ ID NO: 3 or 13 or 27 to 36 having glucoamylaseactivity. In the recombinant yeast host cell of the present disclosure,the first polynucleotide molecule is operatively associated with thesecond polynucleotide molecule. In an embodiment, the heterologousnucleic acid molecules encodes the heterologous polypeptide having theamino acid sequence of SEQ ID NO: 1 or 11, a variant of the amino acidsequence of SEQ ID NO: 1 or 11 having glucoamylase activity, or afragment of the amino acid sequence of SEQ ID NO: 1 or 11 havingglucoamylase activity. In yet another embodiment, the heterologousnucleic acid molecule further comprises a third polynucleotidecomprising a heterologous promoter operatively associated with the firstpolynucleotide and the second polynucleotide allowing the expression ofthe heterologous polypeptide having glucoamylase activity. In anembodiment, the heterologous promoter is capable of allowing theexpression of the heterologous polypeptide having glucoamylase activityduring propagation. In an embodiment, the heterologous polypeptidehaving glucoamylase activity is a secreted polypeptide. In anotherembodiment, the heterologous polypeptide having glucoamylase activity isa membrane-associated polypeptide, such as, for example, a tetheredpolypeptide. In an embodiment, the recombinant yeast host cell comprisesa further heterologous nucleic acid molecule encoding a heterologousalpha-amylase and/or a heterologous glucoamylase. In some specificembodiments, the heterologous alpha-amylase has the amino acid sequenceof any one of SEQ ID NO: 17 to 26, is a variant of the amino acidsequence of any one of SEQ ID NO: 17 to 26 having alpha-amylase activityor is a fragment of the amino acid sequence of any one of SEQ ID NO: 17to 26 having alpha-amylase activity. In some specific embodiments, theheterologous glucoamylase has the amino acid sequence of any one of SEQID NO: 27 to 36, a variant of the amino acid sequence of any one of SEQID NO: 27 to 36 having glucoamylase activity or a fragment of the aminoacid sequence of any one of SEQ ID NO: 27 to 36 having glucoamylaseactivity. In an embodiment, the recombinant yeast host cell is from thegenus Saccharomyces, such as, for example, from the speciesSaccharomyces cerevisiae.

According to a second aspect, the present disclosure provides acomposition comprising the recombinant yeast host cell described hereinand starch.

According to a third aspect, the present disclosure provides a processfor saccharification and fermentation of a biomass into a fermentationproduct, the process comprises contacting the biomass with therecombinant yeast host cell defined herein or the composition definedherein, under a condition that allows the conversion of at least a partof the biomass into the fermentation product (in some embodiments duringa fermentation). In an embodiment, the biomass is derived from orcomprises corn, potato, cassava, rice, wheat, cellulosic material, milo(grain sorghum) or buckwheat. In another embodiment, the biomass isderived from or comprises corn. In still another embodiment, the biomasscomprises or is corn mash. In an embodiment, the fermentation product isethanol. In another embodiment, the conversion/fermentation is conductedin the presence of a stressor. In yet a further embodiment, the stressoris low pH (such as, for example, a pH of 5.0 or lower or a pH of 4.0 orlower). In still a further embodiment, the stressor is an elevatedtemperature. In some embodiments, the process avoids including apurified exogenous enzyme (e.g., achieves 100% enzyme displacement). Insome embodiments, the purified exogenous enzyme is a glucoamylase.

According to a fourth aspect, the present disclosure provides a processfor fermenting a biomass into a fermentation product. The processcomprises contacting the biomass with a recombinant yeast host cell anda reduced dose of a purified exogenous enzyme, under a condition thatallows the conversion of at least a part of the biomass into thefermentation product. The recombinant yeast host cell has a heterologousnucleic acid molecule encoding a heterologous polypeptide having starchor dextrin hydrolase activity, wherein the heterologous nucleic acidmolecule comprises a first polynucleotide encoding a heterologous signalsequence; and a second polynucleotide encoding a heterologouspolypeptide having starch or dextrin hydrolase activity. The firstpolynucleotide molecule is operatively associated with the secondpolynucleotide molecule. The exogenous enzyme is a starch or dextrinhydrolase. The reduced dose of the purified exogenous enzyme is lowerthan a control dose necessary for a control yeast host cell lacking theability to hydrolyze starch or dextrin to complete a correspondingcontrol fermentation. In an embodiment, the control yeast host celllacks the heterologous nucleic acid molecule. In still anotherembodiment, the heterologous polypeptide having starch or dextrinhydrolase activity is an heterologous polypeptide having glucoamylaseactivity. In yet another embodiment, the polypeptide having glucoamylaseactivity has the amino acid sequence of SEQ ID NO: 3, 13 or 27 to 36, isa variant of the amino acid sequence of SEQ ID NO: 3, 13 or 27 to 36having glucoamylase activity, or is a fragment of the amino acidsequence of SEQ ID NO: 3, 13 or 27 to 36 having glucoamylase activity.In still a further embodiment, the heterologous signal sequence has theamino acid sequence of SEQ ID NO: 5, is a variant of the amino acidsequence of SEQ ID NO: 5 having signal sequence activity, or is afragment of the amino acid sequence of SEQ ID NO: 5 having signalsequence activity. In yet another embodiment, the heterologous nucleicacid molecule encodes the heterologous polypeptide having the amino acidsequence of SEQ ID NO: 1 or 11, a variant of the amino acid sequence ofSEQ ID NO: 1 or 11 having glucoamylase activity, or a fragment of theamino acid sequence of SEQ ID NO: 1 or 11 having glucoamylase activity.In still another embodiment, the heterologous nucleic acid moleculefurther comprises a third polynucleotide comprising a heterologouspromoter operatively associated with the first polynucleotide and thesecond polynucleotide allowing the expression of the heterologouspolypeptide having starch or hydrolase activity. In an embodiment, theheterologous promoter is capable of allowing the expression of theheterologous polypeptide having starch or dextrin hydrolase activityduring propagation. In still a further embodiment, the heterologouspolypeptide having starch or dextrin activity is a secreted polypeptide.In yet another embodiment, the heterologous polypeptide having starch ordextrin hydrolase activity is a membrane-associated polypeptide. In yetanother embodiment, the membrane-associated polypeptide is a tetheredpolypeptide. In an embodiment, the recombinant yeast host cell is fromthe genus Saccharomyces and, in a further embodiment, from the speciesSaccharomyces cerevisiae. In yet another embodiment, the biomasscomprises starch or a starch derivative. In still a further embodiment,the biomass is derived from or comprises corn, potato, cassava, rice,wheat, lignocellulosic material or buckwheat. In yet another embodiment,the biomass is derived from or comprises corn. In specific embodimentsthe biomass comprises or is corn mash. In an embodiment, thefermentation product is ethanol. In still another embodiment, thecontrol dose allows achieving a fermentation yield of at least 0.415%,w/v per w/w of biomass. In a further embodiment, the reduced dose allowsachieving a fermentation yield of at least 0.440%, w/v per w/w ofbiomass. In still another embodiment, the reduced dose of the purifiedexogenous enzyme is lower by at least 50%, 55%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%when compared to the control dose. In a specific embodiment, the processexcludes including a purified exogenous enzyme in the biomass prior toor during fermentation. In an embodiment, the reduced dose of theexogenous enzymes is reduced to zero. In still another embodiment, thefermentation yield is equal to or higher than the control fermentationyield of the control fermentation. In yet another embodiment, thefermentation yield is substantially similar to the control fermentationyield of the control fermentation. In some embodiments, the exogenousenzyme is a glucoamylase.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, referencewill now be made to the accompanying drawings, showing by way ofillustration, a preferred embodiment thereof, and in which:

FIG. 1 illustrates the data associated with a corn mash fermentation(analyzed 23 hours into the fermentation). Bars represent midpointethanol titers in fermentation (plotted on the left y axis, in g/L).Lozenges (♦) denote glucose titers and triangles (▴) representyeast-produced (YP) glycerol titers (right y axis, all in g/L). Resultsare shown for strains M2390, M17199, M15621, M23176 and M23177 (seeTable 1 for a description of the strains). The amount (% of controldose) of exogenous glucoamylase used is listed under each bar.

FIG. 2 illustrates the data associated with a corn mash fermentation(analyzed 48 hours into the fermentation). Bars represent endpointethanol titers (plotted on the left y axis, in g/L). Lozenges (♦) denoteglucose titers and triangles (▴) represent yeast-produced (YP) glyceroltiters (right y axis, all in g/L). Results are shown for strains M2390,M17199, M15621, M23176 and M23177 (see Table 1 for a description of thestrains). The amount (% of control dose) of exogenous glucoamylase usedis listed under each bar.

FIG. 3 illustrates the data associated with a permissive corn mashfermentation in the presence or absence of exogenous glucoamylase(analyzed 48 hours into the fermentation). Bars represent endpointethanol titers (plotted on the left y axis, in g/L). Lozenges (♦) denoteglucose titers and triangles (▴) represent yeast-produced (YP) glyceroltiters (right y axis, all in g/L). Results are shown for strains M2390,M17199, M15621 and M23177. The amount (% of control dose) of exogenousglucoamylase used is listed under each bar.

FIG. 4 illustrates the data associated with a non-permissive (lacticacid treatment) corn mash fermentation (analyzed 48 hours into thefermentation). Bars represent endpoint ethanol titers (plotted on theleft y axis, in g/L). Lozenges (♦) denote glucose titers and triangles(▴) represent yeast-produced (YP) glycerol titers (right y axis, all ing/L). Results are shown for strains M2390, M17199, M15621 and M23177 inthe presence or absence of exogenous glucoamylase. The amount (% ofcontrol dose) of exogenous glucoamylase used is listed under each bar.

FIG. 5 illustrates the data associated with a non-permissive (heattreatment) corn mash fermentation (analyzed 48 hours into thefermentation). Bars represent endpoint ethanol titers (plotted on theleft y axis, in g/L). Lozenges (♦) denote glucose titers and triangles(▴) represent yeast-produced (YP) glycerol titers (right y axis, all ing/L). Results are shown for strains M2390, M17199, M15621 and M23177.The amount (% of control dose) of exogenous glucoamylase used is listedunder each bar.

FIG. 6 illustrates the average batch ethanol yield at 60 h fermentationduration (ethanol titer normalized to total solids of slurry in cookingprocess per fermentation batch, results are shown as % w/v per % w/w).The conventional (not genetically modified) yeast (Phase A) was usedwith 100 gal of exogenous glucoamylase cocktail in fermentation, strainM15419 (Phases B and C) was used with 51 gal of exogenous glucoamylasecocktail in fermentation, and strain M23541 (Phases C to K) was usedwith between zero exogenous glucoamylase cocktail in mash propagationand fermentation and 17 gal of exogenous glucoamylase cocktail infermentation. As the total final fermentation durations varied betweenyeast strain types and conditions, data is presented here for allconditions after 60 h of fermentation (60 h following the transfer ofmash propagation to fermentation). Error bars indicate ±1 standarddeviation from the average.

FIG. 7 illustrates the average batch ethanol yield (ethanol titernormalized to total solids of slurry in cooking process per fermentationbatch, results are shown as % w/v per % w/w) at the end of fermentation(fermentation drop). The conventional (not genetically modified) yeast(Phase A) was used with 100 gal of exogenous glucoamylase cocktail infermentation, strain M15419 (Phases B and C) was used with 51 gal ofexogenous glucoamylase cocktail in fermentation, and strain M23541(Phase D to K) was used with between zero exogenous glucoamylasecocktail in mash propagation and fermentation and 17 gal of exogenousglucoamylase cocktail in fermentation. Error bars indicate ±1 standarddeviation from the average.

FIG. 8 illustrates the average batch ethanol yield (ethanol titernormalized to total solids of slurry in cooking process per fermentationbatch, results are shown as % w/v per % w/w) of strain M23541 over thecourse of fermentations. The conventional (not genetically modified)yeast (Phase A) was used with 100 gal of exogenous glucoamylase cocktailin fermentation, strain M15419 (Phases B and C) was used with 51 gal ofexogenous glucoamylase cocktail in fermentation, and strain M23541(Phases D to K) was used with between zero exogenous glucoamylasecocktail in mash propagation and fermentation and 17 gal of exogenousglucoamylase cocktail in fermentation. Error bars indicate ±1 standarddeviation from the average for the conventional (not geneticallymodified) yeast. Letter indicated in legend represents testing phase,and percentage indicated in legend represents percentage displacement ofexogenous GA enzyme cocktail.

FIG. 9 illustrates the data associated with permissive fermentationsanalyzed 24 hours and 52 hours into the fermentation. Bars representendpoint ethanol titers (plotted on the left y axis, in g/L). Resultsare shown for strains Ethanol Red (e.g., active dry yeast or ADY),M23177, M24926 and M23541 in function of the amount (% of dose) ofexogenous glycoamylase used (both listed under each bar).

FIG. 10 illustrates the ethanol titer data of FIG. 9 obtained at 52hours, but plotted as relative ethanol yield compared to Ethanol Red(ADY). For each strain (M23177 ♦, M24926 ● and M23541 ▪) and each enzymeinclusion, the drop ethanol titer is plotted as a ratio to the dropethanol titer of Ethanol Red (ADY) with 100% enzyme inclusion (y axis).The exogenous enzyme inclusion (% of dose) for each condition isreported as a percentage from the dose used with ADY and is indicated onthe x axis.

DETAILED DESCRIPTION

As shown herein, yeast strains genetically engineered to secrete astarch or dextrin hydrolase (such as for example a glucoamylase) canmake a fermentation product (such as ethanol) using reduced levels(e.g., a reduced dose) of exogenous enzyme (which may be present in theform of a cocktail) to complete the fermentation.

In dry mill fuel ethanol production facilities, corn milling, themashing or cooking process, and finished fermentation beer distillationand product recovery unit operations are carried out continuously. Infacilities utilizing batch fermentation, the liquefact mash is fermentedin batches (or, more specifically, fed-batches) in the otherwisecontinuous ethanol production process. Facilities utilizing batchfermentation have multiple fermentation vessels (“fermentors”, typicallybetween 4 and 7 vessels for facilities as originally built) operated inparallel. The fermentors are operated in a cyclical mode such that atany point in time one fermentor is being filled with liquefact, thefinished fermentation beer from another fermentor is being transferredto the holding vessel prior to distillation (the “beer well”) and theemptied fermentor cleaned, and the remaining fermentors are in variousstages of fermentation (measured by duration following the start of fillof the fermentor with liquefact). The duration of one cycle offermentors is set by the total number of fermentors in use at theproduction facility, the working volume of the fermentors (the totalvolume to which fermentors are filled with liquefact, transfer of mashpropagation or direct pitch yeast addition, exogenous GA enzyme cocktailand other added fermentation enzymes, nitrogen source addition, and anyother fermentation ingredients), and the volumetric flow rate ofliquefact discharged from the mashing process at the facility. Onefermentation batch occurs during the period between when liquefactbegins to be added to a fermentor during a cycle and when the finishedfermentation beer begins to be transferred to the beer well (the timereferred to as fermentation “drop” or the “end of fermentation”) duringthe same cycle. The maximum duration of a fermentation batch at aproduction facility is one cycle duration minus the time required toempty and clean the fermentor following the batch in order to preparefor the next cycle.

The present disclosure also provides recombinant yeast host cells thatcan be used under conditions of saccharification and fermentation of abiomass. Glucoamylase are usually secreted by the cell expressing a geneencoding same. Most glucoamylase sequences include a signal sequencewhich enables or facilitates the secretion of the enzyme. In the presentdisclosure, it has been recognized that using a signal sequence from theSaccharomyces cerevisiae alpha-mating factor 1 (e.g., having the aminoacid sequence of SEQ ID NO: 5, a variant thereof or a fragment thereof)allows the displacement, at least in part, of exogenous (purified)glucoamylase during the fermentation process. In some additionalembodiments, using a signal sequence from the Saccharomyces cerevisiaealpha-mating factor 1 (e.g., having the amino acid sequence of SEQ IDNO: 5, a variant thereof or a fragment thereof) increased the robustnessof the recombinant yeast host cell expressing same.

Recombinant Yeast Host Cell

The heterologous polypeptides having starch or dextrin hydrolaseactivity (such as, for example, glucoamylases) are expressed in arecombinant yeast host cell. As used in the context of the presentdisclosure, a heterologous polypeptide having starch or dextrinhydrolase activity is a polypeptide capable of cleaving a starch and/ora dextrin molecule into smaller fragments. As such, the recombinantyeast host cell of the present disclosure includes at least one geneticmodification. In the context of the present disclosure, when recombinantyeast cell is qualified has “having a genetic modification” or as being“genetically engineered”, it is understood to mean that it has beenmanipulated to either add at least one or more heterologous or exogenousnucleic acid residue and/or remove at least one endogenous (or native)nucleic acid residue. The genetic manipulation(s) did not occur innature and is the results of in vitro manipulations of the recombinanthost cell. When the genetic modification is the addition of aheterologous nucleic acid molecule, such addition can be made once ormultiple times at the same or different integration sites. When thegenetic modification is the modification of an endogenous nucleic acidmolecule, it can be made in one or both copies of the targetedgene/non-coding region. In a specific embodiment, the recombinant yeasthost cell having the genetic modification has a heterologous nucleicacid molecule encoding a heterologous polypeptide having glucoamylaseactivity.

When expressed in a recombinant yeast host cell, the heterologouspolypeptide (such as those having glucoamylase activity, e.g. aglucoamylase) described herein are encoded on one or more heterologousnucleic acid molecule. In some embodiments, heterologous polypeptidedescribed herein can be encoded on one heterologous nucleic acidmolecule, two heterologous nucleic acid molecules or copies, threeheterologous nucleic acid molecules or copies, four heterologous nucleicacid molecules or copies, five heterologous nucleic acid molecules orcopies, six heterologous nucleic acid molecules or copies, sevenheterologous nucleic acid molecules or copies, or eight or moreheterologous nucleic acid molecules or copies. The term “heterologous”when used in reference to a nucleic acid molecule (such as a promoter ora coding sequence) refers to a nucleic acid molecule that is notnatively found in the recombinant yeast host cell. “Heterologous” alsoincludes a native coding region, or portion thereof, that was removedfrom the organism (which can, in some embodiments, be a source organism)and subsequently reintroduced into the organism in a form that isdifferent from the corresponding native gene, e.g., not in its naturallocation in the organism's genome. The heterologous nucleic acidmolecule is purposively introduced into the recombinant yeast host cell.The term “heterologous” as used herein also refers to an element(nucleic acid or polypeptide) that is derived from a source other thanthe endogenous source. Thus, for example, a heterologous element couldbe derived from a different strain of host cell, or from an organism ofa different taxonomic group (e.g., different kingdom, phylum, class,order, family genus, or species, or any subgroup within one of theseclassifications).

When a heterologous nucleic acid molecule is present in the recombinantyeast host cell, it can be integrated in the host cell's chromosome. Theterm “integrated” as used herein refers to genetic elements that areplaced, through molecular biology techniques, into the chromosome(s) ofthe recombinant yeast host cell. For example, genetic elements can beplaced into the chromosome(s) of the host cell as opposed to in a vectorsuch as a plasmid carried by the host cell. Methods for integratinggenetic elements into the chromosome(s) of a host cell are well known inthe art and include homologous recombination. The heterologous nucleicacid molecule can be present in one or more copies in the yeast hostcell's chromosome(s). Alternatively, the heterologous nucleic acidmolecule can be independently replicating from the yeast's chromosome.In such embodiment, the nucleic acid molecule can be stable andself-replicating.

In the context of the present disclosure, the yeast host cell can be arecombinant yeast host cell. Suitable yeast host cells can be, forexample, from the genus Saccharomyces, Kluyveromyces, Arxula,Debaryomyces, Candida, Pichia, Phaffia, Schizosaccharomyces, Hansenula,Kloeckera, Schwanniomyces or Yarrowia. Suitable yeast species caninclude, for example, Saccharomyces cerevisiae, Saccharomyces bulderi,Saccharomyces barnetti, Saccharomyces exiguus, Saccharomyces uvarum,Saccharomyces diastaticus, Kluyveromyces lactis, Kluyveromyces marxianusor Kluyveromyces fragilis. In some embodiments, the yeast is selectedfrom the group consisting of Saccharomyces cerevisiae,Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichiastipitis (Komagatella phaffi), Yarrowia lipolytica, Hansenulapolymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans,Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomycespombe and Schwanniomyces occidentalis. In one particular embodiment, theyeast is Saccharomyces cerevisiae. In some embodiments, the host cellcan be an oleaginous yeast cell. For example, the oleaginous yeast hostcell can be from the genus Blakeslea, Candida, Cryptococcus,Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium,Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. In somealternative embodiments, the host cell can be an oleaginous microalgaehost cell (e.g., for example, from the genus Thraustochytrium orSchizochytrium). In an embodiment, the recombinant yeast host cell isfrom the genus Saccharomyces and, in some additional embodiments, fromthe species Saccharomyces cerevisiae.

In some embodiments, the nucleic acid molecules encoding theheterologous polypeptides, fragments or variants that can be introducedinto the recombinant yeast host cells are codon-optimized with respectto the intended recipient recombinant yeast host cell. As used hereinthe term “codon-optimized coding region” means a nucleic acid codingregion that has been adapted for expression in the cells of a givenorganism by replacing at least one, or more than one, codons with one ormore codons that are more frequently used in the genes of that organism.In general, highly expressed genes in an organism are biased towardscodons that are recognized by the most abundant tRNA species in thatorganism. One measure of this bias is the “codon adaptation index” or“CAI,” which measures the extent to which the codons used to encode eachamino acid in a particular gene are those which occur most frequently ina reference set of highly expressed genes from an organism. The CAI ofcodon optimized heterologous nucleic acid molecule described hereincorresponds to between about 0.8 and 1.0, between about 0.8 and 0.9, orabout 1.0.

The heterologous nucleic acid molecules of the present disclosure cancomprise a coding region for the heterologous polypeptide. A DNA or RNA“coding region” is a DNA or RNA molecule which is transcribed and/ortranslated into a polypeptide in a cell in vitro or in vivo when placedunder the control of appropriate regulatory sequences. “Suitableregulatory regions” refer to nucleic acid regions located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding region, and which influence the transcription, RNAprocessing or stability, or translation of the associated coding region.Regulatory regions may include promoters, translation leader sequences,RNA processing site, effector binding site and stem-loop structure. Theboundaries of the coding region are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding region can include, but is not limited to,prokaryotic regions, cDNA from mRNA, genomic DNA molecules, syntheticDNA molecules, or RNA molecules. If the coding region is intended forexpression in a eukaryotic cell, a polyadenylation signal andtranscription termination sequence will usually be located 3′ to thecoding region. In an embodiment, the coding region can be referred to asan open reading frame. “Open reading frame” is abbreviated ORF and meansa length of nucleic acid, either DNA, cDNA or RNA, that comprises atranslation start signal or initiation codon, such as an ATG or AUG, anda termination codon and can be potentially translated into a polypeptidesequence.

The nucleic acid molecules described herein can comprise transcriptionaland/or translational control regions. “Transcriptional and translationalcontrol regions” are DNA regulatory regions, such as promoters,enhancers, terminators, and the like, that provide for the expression ofa coding region in a host cell. In eukaryotic cells, polyadenylationsignals are control regions.

The heterologous nucleic acid molecule can be introduced in the hostcell using a vector. A “vector,” e.g., a “plasmid”, “cosmid” or“artificial chromosome” (such as, for example, a yeast artificialchromosome) refers to an extra chromosomal element and is usually in theform of a circular double-stranded DNA molecule. Such vectors may beautonomously replicating sequences, genome integrating sequences, phageor nucleotide sequences, linear, circular, or supercoiled, of a single-or double-stranded DNA or RNA, derived from any source, in which anumber of nucleotide sequences have been joined or recombined into aunique construction which is capable of introducing a promoter fragmentand DNA sequence for a selected gene product along with appropriate 3′untranslated sequence into a cell.

Heterologous Polypeptides Having Starch or Dextrin Hydrolase Activityand Nucleic Acid Molecules Encoding Same

The heterologous nucleic acid molecule present in the recombinant yeasthost cell includes a heterologous polynucleotide encoding a signalsequence. As it is known in the art, a signal sequence corresponds to ashort stretch of amino acid residues (usually no longer than 50contiguous amino acids and usually located at the amino terminus of thepolypeptide) which are capable of guiding the remainder of thepolypeptide for secretion. The signal sequence is usually cleaved uponthe secretion of the polypeptide and thus is not necessarily involvedwith the enzymatic activity of the secreted polypeptide (e.g.,glucoamylase activity in the present disclosure). In embodiments, thesignal sequence encoded by the heterologous nucleic acid molecule (whichcan be associated with the heterologous polypeptide having glucoamylaseactivity) can have the amino acid sequence of SEQ ID NO: 5 or of thesection spanning residues 1 to 21 of SEQ ID NO: 17, 1 to 21 of SEQ IDNO: 18, 1 to 23 of SEQ ID NO: 19, 1 to 19 of SEQ ID NO: 20, 1 to 25 ofSEQ ID NO: 21, 1 to 22 of SEQ ID NO: 22, 1 to 29 of SEQ ID NO: 23, 1 to16 of SEQ ID NO: 24, 1 to 23 of SEQ ID NO: 25, 1 to 21 of SEQ ID NO: 26,1 to 17 of SEQ ID NO: 27, 1 to 20 of SEQ ID NO: 28, 1 to 22 of SEQ IDNO: 29, 1 to 18 of SEQ ID NO: 30, 1 to 25 of SEQ ID NO: 31, 1 to 19 ofSEQ ID NO: 32, 1 to 18 of SEQ ID NO: 33, 1 to 19 of SEQ ID NO: 34, 1 to18 of SEQ ID NO: 35, 1 to 18 of SEQ ID NO: 36 as well as variants andfragments thereof. In embodiments in which the heterologous polypeptidehaving glucoamylase activity has the amino acid sequence of SEQ ID NO: 3or 13, the signal sequence encoded by the heterologous nucleic acidmolecule (which can be associated with the heterologous polypeptidehaving glucoamylase activity) can have the amino acid sequence of SEQ IDNO: 5, a variant thereof or a fragment thereof.

The first polynucleotide can encode a signal sequence, a variant of asignal sequence having signal sequence activity or a fragment of asignal sequence having signal sequence activity. A variant signalsequence comprises at least one amino acid difference when compared tothe amino acid sequence of the native or wild-type signal sequence andexhibits a biological activity substantially similar to the native(wild-type) signal sequence (e.g., the ability to guide the heterologouspolypeptide having glucoamylase activity for secretion). The signalsequence “variants” have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological activity whencompared to the wild-type signal sequence described herein. The signalsequence “variants” have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the wild-type signalsequence described herein. The level of identity can be determinedconventionally using known computer programs. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in: Computational Molecular Biology (Lesk, A. M., ed.) OxfordUniversity Press, N Y (1988); Biocomputing: Informatics and GenomeProjects (Smith, D. W., ed.) Academic Press, N Y (1993); ComputerAnalysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G.,eds.) Humana Press, N J (1994); Sequence Analysis in Molecular Biology(von Heinje, G., ed.) Academic Press (1987); and Sequence AnalysisPrimer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991).Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignments of thesequences disclosed herein were performed using the Clustal method ofalignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLB 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The variant signal sequence described herein may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group. A “variant” of thewild-type signal sequence can be a conservative variant or an allelicvariant. As used herein, a conservative variant refers to alterations inthe amino acid sequence that do not adversely affect the biologicalfunctions of the signal sequence. A substitution, insertion or deletionis said to adversely affect the signal sequence when the alteredsequence prevents or disrupts a biological function associated with thesignal sequence. For example, the overall charge, structure orhydrophobic-hydrophilic properties of the signal sequence can be alteredwithout adversely affecting a biological activity. Accordingly, theamino acid sequence can be altered, for example to render the signalsequence more hydrophobic or hydrophilic, without adversely affectingthe biological activities of the signal sequence.

The signal sequence can be a fragment of the signal sequence or afragment of a variant signal sequence. A signal sequence fragmentcomprises at least one less amino acid residue when compared to theamino acid sequence of the full length signal sequence or variantpossesses and still possess a biological activity substantially similarto the native full-length signal sequence or variant. The signalsequence “fragments” have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological activity whencompared to the full-length signal sequence or variants describedherein. Signal sequence “fragments” have at least at least 2, 3, 4, 5,6, 7, 8, 9, 10 or more consecutive amino acids of the full-length signalsequence or variants described herein. The signal sequence “fragments”can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identity to the full-length signal sequence or variantsdescribed herein.

The heterologous nucleic acid molecule of the present disclosure alsoincludes a second polynucleotide encoding the heterologous polypeptidehaving starch or dextrin hydrolase activity (such as an heterologouspolypeptide having glucoamylase activity). In the context of the presentdisclosure, the first and second polynucleotides are in frame andoperatively associated so as to encode a single polypeptide (which isintended to be cleaved so as to release the polypeptide havingglucoamylase activity upon the secretion of the single polypeptide). Inthe heterologous nucleic acid molecule, the first polynucleotide islocated upstream (5′) with respect to the second polynucleotide.Otherwise stated, the second polynucleotide is located downstream (3′)with respect to the first polynucleotide in the heterologous nucleicacid sequence.

As indicated above, the recombinant yeast host cell can bear a geneticmodification for expressing at least one heterologous polypeptide havingstarch or dextrin hydrolase activity (such as a heterologous polypeptidehaving glucoamylase activity). In some embodiments, the recombinantyeast host cell is obtained by introducing one or more heterologousnucleic acid molecule encoding one or more of the heterologouspolypeptide in the recombinant yeast host cell. In some embodiments, thegenetic modification(s) in the recombinant yeast host cell of thepresent disclosure comprise or consist essentially of or consist ofexpressing a heterologous polypeptide having starch or dextrin hydrolaseactivity. In the context of the present disclosure, the expression “thegenetic modification in the recombinant yeast host consist essentiallyof a genetic modification for expressing a heterologous polypeptidehaving starch digesting glucoamylase activity” refers to the fact thatthe recombinant yeast host cell only includes this genetic modificationto modulate the expression of a polypeptide having starch digestingglucoamylase activity levels but can nevertheless include other geneticmodifications which are unrelated to the expression of a glucoamylase(native or heterologous).

As indicated above, the heterologous polypeptide is a polypeptide havingstarch or dextrin digesting activity. As used herein, a polypeptidehaving glucoamylase activity refers to a polypeptide having the abilityto hydrolyze starch (which can have been heat-treated) directly toglucose. For example, a polypeptide having glucoamylase activity maycomprise a catalytic domain and a starch binding domain. The catalyticdomain and the starch binding domain may be connected by a connectingloop or linker. In some alternative embodiments, the polypeptides havingglucoamylase activity can be derived from a fungus, for example, fromthe genus Rasamsonia (sometimes known or referred to as Talaromyces)and, in some instances, from the species Rasamsonia emersonii (sometimesknown or referred to as Talaromyces emersonii). In some specificembodiments, the heterologous polypeptide having starch digestingglucoamylase activity can have the amino acid sequence of SEQ ID NO: 3(which refers to Uniprot Q9C1V4), be a variant of the amino acidsequence of SEQ ID NO: 3 (having glucoamylase activity) or be a fragmentof the amino acid sequence of SEQ ID NO: 3 (having glucoamylaseactivity). In yet another specific embodiment, the heterologous nucleicacid molecule can comprise the nucleic acid sequence of SEQ ID NO: 4, bea variant of the nucleic acid sequence of SEQ ID NO: 4 (encoding aglucoamylase) or be a fragment of the nucleic acid sequence of SEQ IDNO: 4 (encoding a glucoamylase). In another specific embodiment, theheterologous polypeptide having glucoamylase activity can have the aminoacid sequence of SEQ ID NO: 1, be a variant of the amino acid sequenceof SEQ ID NO: 1 (having glucoamylase activity) or be a fragment of theamino acid sequence of SEQ ID NO: 1 (having glucoamylase activity). Inyet another specific embodiment, the heterologous nucleic acid moleculecan comprise the nucleic acid sequence of SEQ ID NO: 2, be a variant ofthe nucleic acid sequence of SEQ ID NO: 2 (encoding a glucoamylase) orbe a fragment of the nucleic acid sequence of SEQ ID NO: 2 (encoding aglucoamylase). In some specific embodiments, the heterologouspolypeptide having starch digesting glucoamylase activity can have theamino acid sequence of SEQ ID NO: 13 (which refers to UniprotA0A0F4YWQ6), be a variant of the amino acid sequence of SEQ ID NO: 13(having glucoamylase activity) or be a fragment of the amino acidsequence of SEQ ID NO: 13 (having glucoamylase activity). In yet anotherspecific embodiment, the heterologous nucleic acid molecule can comprisethe nucleic acid sequence of SEQ ID NO: 14, be a variant of the nucleicacid sequence of SEQ ID NO: 14 (encoding a glucoamylase) or be afragment of the nucleic acid sequence of SEQ ID NO: 14 (encoding aglucoamylase). In another specific embodiment, the heterologouspolypeptide having glucoamylase activity can have the amino acidsequence of SEQ ID NO: 11, be a variant of the amino acid sequence ofSEQ ID NO: 11 (having glucoamylase activity) or be a fragment of theamino acid sequence of SEQ ID NO: 11 (having glucoamylase activity). Inyet another specific embodiment, the heterologous nucleic acid moleculecan comprise the nucleic acid sequence of SEQ ID NO: 12, be a variant ofthe nucleic acid sequence of SEQ ID NO: 12 (encoding a glucoamylase) orbe a fragment of the nucleic acid sequence of SEQ ID NO: 12 (encoding aglucoamylase).

In some further embodiments, the recombinant yeast host cell can includea further genetic modification (which can be the introduction of afurther heterologous nucleic acid molecule) for expressing a furtherheterologous glucoamylase (e.g., different from the R. emersoniiglucoamylase described above). For example, the further heterologousglucoamylase can be from a Gloeophyllum sp., such as, for example, fromGloeophyllum trabeum. In an embodiment, the further heterologousglucoamylase corresponds to Uniprot S7Q4V9 or GenBank AccessionNumber_007866834. In another embodiment, the further heterologousglucoamylase can have the amino acid sequence of SEQ ID NO: 27, be avariant of the amino acid sequence of SEQ ID NO: 27 having glucoamylaseactivity or be a fragment of the amino acid sequence of SEQ ID NO: 27having glucoamylase activity (which can, in an embodiment, correspond toa fragment of the amino acid sequence of SEQ ID NO: 27 lacking itssignal sequence, e.g., for example between residues 18 to 576 of SEQ IDNO: 27). For example, the further heterologous glucoamylase can be froma Trichoderma sp., such as, for example, from Trichoderma reesii. In anembodiment, the further heterologous glucoamylase corresponds to UniprotG0R866 or GenBank Accession Number_XP 006960925. In another embodiment,the further heterologous glucoamylase can have the amino acid sequenceof SEQ ID NO: 28, be a variant of the amino acid sequence of SEQ ID NO:28 having glucoamylase activity or be a fragment of the amino acidsequence of SEQ ID NO: 28 having glucoamylase activity (which can, in anembodiment, correspond to a fragment of the amino acid sequence of SEQID NO: 28 lacking its signal sequence, e.g., for example betweenresidues 21 to 632 of SEQ ID NO: 28). For example, the furtherheterologous glucoamylase can be from a Trametes sp., such as, forexample, from Trametes cingulata. In another embodiment, the furtherheterologous glucoamylase can have the amino acid sequence of SEQ ID NO:29, be a variant of the amino acid sequence of SEQ ID NO: 29 havingglucoamylase activity or be a fragment of the amino acid sequence of SEQID NO: 29 having glucoamylase activity (which can, in an embodiment,correspond to a fragment of the amino acid sequence of SEQ ID NO: 29lacking its signal sequence, e.g., for example between residues 23 to574 of SEQ ID NO: 29). For example, the further heterologousglucoamylase can be from a Athelia sp., such as, for example, fromAthelia rolfsil. In an embodiment, the further heterologous glucoamylasecorresponds to Uniprot Q12596 or GenBank Accession Number_BAA08436. Inanother embodiment, the further heterologous glucoamylase can have theamino acid sequence of SEQ ID NO: 30, be a variant of the amino acidsequence of SEQ ID NO: 30 having glucoamylase activity or be a fragmentof the amino acid sequence of SEQ ID NO: 30 having glucoamylase activity(which can, in an embodiment, correspond to a fragment of the amino acidsequence of SEQ ID NO: 30 lacking its signal sequence, e.g., for examplebetween residues 19 to 579 of SEQ ID NO: 30). For example, the furtherheterologous glucoamylase can be from a Rhizopus sp., such as, forexample, from Rhizopus oryzae. In an embodiment, the furtherheterologous glucoamylase corresponds to Uniprot P07683 or GenBankAccession Number P07683. In another embodiment, the further heterologousglucoamylase can have the amino acid sequence of SEQ ID NO: 31, be avariant of the amino acid sequence of SEQ ID NO: 31 having glucoamylaseactivity or be a fragment of the amino acid sequence of SEQ ID NO: 31having glucoamylase activity (which can, in an embodiment, correspond toa fragment of the amino acid sequence of SEQ ID NO: 31 lacking itssignal sequence, e.g., for example, between residues 26 and 604 of SEQID NO: 31). For example, the further heterologous glucoamylase can befrom a Aspergillus sp., such as, for example, from Aspergillus oryzae.In an embodiment, the further heterologous glucoamylase corresponds toUniprot P36914 or GenBank Accession Number BAA00841. In anotherembodiment, the further heterologous glucoamylase can have the aminoacid sequence of SEQ ID NO: 32, be a variant of the amino acid sequenceof SEQ ID NO: 32 having glucoamylase activity or be a fragment of theamino acid sequence of SEQ ID NO: 32 having glucoamylase activity (whichcan, in an embodiment, correspond to a fragment of the amino acidsequence of SEQ ID NO: 32 lacking its signal sequence, e.g., for examplebetween residues 20 to 612 of SEQ ID NO: 32). In yet another example,the further heterologous glucoamylase can be from Aspergillus awamori.In an embodiment, the further heterologous glucoamylase corresponds toUniprot Q76L97 or GenBank Accession Number BAD06004. In anotherembodiment, the further heterologous glucoamylase can have the aminoacid sequence of SEQ ID NO: 35, be a variant of the amino acid sequenceof SEQ ID NO: 35 having glucoamylase activity or be a fragment of theamino acid sequence of SEQ ID NO: 35 having glucoamylase activity (whichcan, in an embodiment, correspond to a fragment of the amino acidsequence of SEQ ID NO: 35 lacking its signal sequence, e.g., for examplebetween residues 19 to 639 of SEQ ID NO: 35). In yet another example,the further heterologous glucoamylase can be from Aspergillus niger. Inan embodiment, the further heterologous glucoamylase corresponds toUniprot Q870G8 or GenBank Accession Number AAP04499. In anotherembodiment, the further heterologous glucoamylase can have the aminoacid sequence of SEQ ID NO: 36, be a variant of the amino acid sequenceof SEQ ID NO: 36 having glucoamylase activity or be a fragment of theamino acid sequence of SEQ ID NO: 36 having glucoamylase activity (whichcan, in an embodiment, correspond to a fragment of the amino acidsequence of SEQ ID NO: 36 lacking its signal sequence, e.g., for examplebetween residues 19 to 639 of SEQ ID NO: 36). For example, the furtherheterologous glucoamylase can be from a Ophiostoma sp., such as, forexample, from Ophiostoma floccosum. In an embodiment, the furtherheterologous glucoamylase corresponds to Uniprot Q06SN2 or GenBankAccession Number ABF72529. In another embodiment, the furtherheterologous glucoamylase can have the amino acid sequence of SEQ ID NO:33, be a variant of the amino acid sequence of SEQ ID NO: 33 havingglucoamylase activity or be a fragment of the amino acid sequence of SEQID NO: 33 having glucoamylase activity (which can, in an embodiment,correspond to a fragment of the amino acid sequence of SEQ ID NO: 33lacking its signal sequence, e.g., for example between residues 19 to630 of SEQ ID NO: 33). For example, the further heterologousglucoamylase can be from a Trichocladium sp., such as, for example, fromTrichocladium griseum. In an embodiment, the further heterologousglucoamylase corresponds to Uniprot Q12623 or GenBank Accession NumberAAA33386. In another embodiment, the further heterologous glucoamylasecan have the amino acid sequence of SEQ ID NO: 34, be a variant of theamino acid sequence of SEQ ID NO: 34 having glucoamylase activity or bea fragment of the amino acid sequence of SEQ ID NO: 34 havingglucoamylase activity (which can, in an embodiment, correspond to afragment of the amino acid sequence of SEQ ID NO: 34 lacking its signalsequence, e.g., for example between residues 20 and 620 of SEQ ID NO:34).

A variant glucoamylase comprises at least one amino acid difference(substitution or addition) when compared to the amino acid sequence ofthe glucoamylase polypeptide of SEQ ID NO: 1, 3, 11, 13 or 27 to 36 andstill exhibits glucoamylase activity. In an embodiment, the variantglucoamylase exhibits at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of theactivity of the native or wild-type glucoamylase having the amino acidsequence of SEQ ID NO: 1, 3, 11, 13 or 27 to 36. The glucoamylasevariants also have at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identitywhen compared to the wild-type or native glucoamylase having the aminoacid sequence of SEQ ID NO: 1, 3, 11, 13 or 27 to 36 over its entirelength. The term “percent identity”, as known in the art, is arelationship between two or more polypeptide sequences, as determined bycomparing the sequences. The level of identity can be determinedconventionally using known computer programs. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in: Computational Molecular Biology (Lesk, A. M., ed.) OxfordUniversity Press, N Y (1988); Biocomputing: Informatics and GenomeProjects (Smith, D. W., ed.) Academic Press, N Y (1993); ComputerAnalysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G.,eds.) Humana Press, N J (1994); Sequence Analysis in Molecular Biology(von Heinje, G., ed.) Academic Press (1987); and Sequence AnalysisPrimer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991).Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignments of thesequences disclosed herein were performed using the Clustal method ofalignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLB 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The variant glucoamylases described herein may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide for purification of the polypeptide.Conservative substitutions typically include the substitution of oneamino acid for another with similar characteristics, e.g., substitutionswithin the following groups: valine, glycine; glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. Other conservative amino acid substitutions are known in theart and are included herein. Non-conservative substitutions, such asreplacing a basic amino acid with a hydrophobic one, are also well-knownin the art.

A variant glucoamylase can also be a conservative variant or an allelicvariant. As used herein, a conservative variant refers to alterations inthe amino acid sequence that do not adversely affect the biologicalfunctions of the starch digesting glucoamylase. A substitution,insertion or deletion is said to adversely affect the polypeptide whenthe altered sequence prevents or disrupts a biological functionassociated with the starch digesting glucoamylase (e.g., the hydrolysisof starch into glucose). For example, the overall charge, structure orhydrophobic-hydrophilic properties of the polypeptide can be alteredwithout adversely affecting a biological activity. Accordingly, theamino acid sequence can be altered, for example to render the peptidemore hydrophobic or hydrophilic, without adversely affecting thebiological activities of the starch digesting glucoamylase.

The present disclosure also provide fragments of the glucoamylase andglucoamylase variants described herein. A fragment comprises at leastone less amino acid residue when compared to the amino acid sequence ofthe catalytic domain or the glucoamylase polypeptide or variant andstill possess the enzymatic activity of the full-length glucoamylase. Inan embodiment, the glucoamylase fragment exhibits at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% activity when compared to the full-lengthglucoamylase having the amino acid of SEQ ID NO: 1, 3, 11, 13 or 27 to36 or variants thereof. The glucoamylase fragments can also have atleast 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity when compared to theglucoamylase having the amino acid sequence of SEQ ID NO: 1, 3, 11, 13or 27 to 36 or variants thereof. The fragment can be, for example, atruncation of one or more amino acid residues at the amino-terminus, thecarboxy terminus or both termini of the starch digesting glucoamylasepolypeptide or variant. In a specific embodiment, the fragmentcorresponds to a polypeptide of any one of SEQ ID NO: 27 to 36 to whichthe signal sequence has been removed. Alternatively or in combination,the fragment can be generated from removing one or more internal aminoacid residues. In an embodiment, the glucoamylase fragment has at least100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or moreconsecutive amino acids of the glucoamylase having the amino acidsequence of SEQ ID NO: 1, 3, 11, 13 or 27 to 36 or variants thereof.

The heterologous polypeptides having starch or dextrin hydrolaseactivity described herein are secreted polypeptides. In someembodiments, secreted heterologous polypeptides having starch or dextrinhydrolase activity are released in the culture/fermentation medium anddo not remain physically attached to the recombinant yeast host cell. Inalternative embodiments, the heterologous starch or dextrin hydrolase ofthe present disclosure can be secreted, but they remain physicallyassociated with the recombinant yeast host cell. In an embodiment, atleast one portion (usually at least one terminus) of the heterologousstarch or dextrin hydrolase is bound, covalently, non-covalently and/orelectrostatically for example, to the cell wall (and in some embodimentsto the cytoplasmic membrane) of the recombinant yeast host cell. Forexample, the heterologous starch or dextrin hydrolase can be modified tobear one or more transmembrane domains, to have one or more lipidmodifications (myristoylation, palmitoylation, farnesylation and/orprenylation), to interact with one or more membrane-associatedpolypeptide and/or to interactions with the cellular lipid rafts. Whilethe heterologous starch or dextrin hydrolases may not be directly boundto the cell membrane or cell wall (e.g., such as when binding occurs viaa tethering moiety), the polypeptide is nonetheless considered a“cell-associated” heterologous polypeptide according to the presentdisclosure.

In some embodiments, the heterologous starch or dextrin hydrolase can beexpressed to be located at and associated to the cell wall of therecombinant yeast host cell. In some embodiments, the heterologousstarch or dextrin hydrolase is expressed to be located at and associatedto the external surface of the cell wall of the host cell. Recombinantyeast host cells all have a cell wall (which includes a cytoplasmicmembrane) defining the intracellular (e.g., internally-facing thenucleus) and extracellular (e.g., externally-facing) environments. Theheterologous starch or dextrin hydrolase can be located at (and in someembodiments, physically associated to) the external face of therecombinant yeast host's cell wall and, in further embodiments, to theexternal face of the recombinant yeast host's cytoplasmic membrane. Inthe context of the present disclosure, the expression “associated to theexternal face of the cell wall/cytoplasmic membrane of the recombinantyeast host cell” refers to the ability of the heterologous starch ordextrin hydrolase to physically integrate (in a covalent or non-covalentfashion), at least in part, in the cell wall (and in some embodiments inthe cytoplasmic membrane) of the recombinant yeast host cell. Thephysical integration can be attributed to the presence of, for example,a transmembrane domain on the heterologous polypeptide, a domain capableof interacting with a cytoplasmic membrane polypeptide on theheterologous polypeptide, a post-translational modification made to theheterologous polypeptide (e.g., lipidation), etc.

In some circumstances, it may be warranted to increase or provide cellassociation to some heterologous starch or dextrin hydrolases becausethey exhibit insufficient intrinsic cell association or simply lackintrinsic cell association. In such embodiment, it is possible toprovide the heterologous starch or dextrin hydrolase as a chimericconstruct by combining it with a tethering amino acid moiety which willprovide or increase attachment to the cell wall of the recombinant yeasthost cell. In such embodiment, the chimeric heterologous polypeptidewill be considered “tethered”. It is preferred that the amino acidtethering moiety of the chimeric polypeptide be neutral with respect tothe biological activity of the heterologous starch or dextrin hydrolase,e.g., does not interfere with the biological activity (such as, forexample, the enzymatic activity) of the heterologous starch or dextrinhydrolase. In some embodiments, the association of the amino acidtethering moiety with the heterologous starch or dextrin hydrolase canincrease the biological activity of the heterologous polypeptide (whencompared to the non-tethered, “free” form).

In an embodiment, a tethering moiety can be used to be expressed withthe heterologous starch or dextrin hydrolase to locate the heterologouspolypeptide to the wall of the recombinant yeast host cell. Varioustethering amino acid moieties are known art and can be used in thechimeric polypeptides of the present disclosure. The tethering moietycan be a transmembrane domain found on another polypeptide and allow thechimeric polypeptide to have a transmembrane domain. In such embodiment,the tethering moiety can be derived from the FLO1 polypeptide. In stillanother example, the amino acid tethering moiety can be modifiedpost-translation to include a glycosylphosphatidylinositol (GPI) anchorand allow the chimeric polypeptide to have a GPI anchor. GPI anchors areglycolipids attached to the terminus of a polypeptide (and in someembodiments, to the carboxyl terminus of a polypeptide) which allows theanchoring of the polypeptide to the cytoplasmic membrane of the cellmembrane. Tethering amino acid moieties capable of providing a GPIanchor include, but are not limited to those associated with/derivedfrom a SED1 polypeptide, a TIR1 polypeptide, a CWP2 polypeptide, a CCW12polypeptide, a SPI1 polypeptide, a PST1 polypeptide or a combination ofa AGA1 and a AGA2 polypeptide. In an embodiment, the tethering moietyprovides a GPI anchor and, in still a further embodiment, the tetheringmoiety is derived from the SPI1 polypeptide or the CCW12 polypeptide.

The tethering amino acid moiety can be a variant of a known/nativetethering amino acid moiety. The tethering amino acid moiety can be afragment of a known/native tethering amino acid moiety or fragment of avariant of a known/native tethering amino acid moiety.

In embodiments in which an amino acid tethering moiety and/or signalsequence may be desirable, the heterologous polypeptide can be providedas a chimeric polypeptide expressed by the recombinant yeast host celland having one of the following formulae:

(NH₂)SS-HP-L-TT(COOH)  (I) or

(NH₂)SS-TT-L-HP(COOH)  (II)

In both of these formulae, the residue “HP” refers to a heterologouspolypeptide moiety, the residue “SS” refers to the signal sequence(which cannot have the amino acid sequence of SEQ ID NO: 5), the residue“L” refers to the presence of an optional linker, and the residue “TT”refers to an optional amino acid tethering moiety. In the chimericpolypeptides of formula (I), the amino (NH₂ or N) terminus of the aminoacid tether is located (directly or indirectly) at the carboxyl (COOH orC) terminus of the heterologous starch or dextrin hydrolase moiety. Inthe chimeric polypeptides of formula (I), the amino (NH₂ or N) terminusof the heterologous starch or dextrin hydrolase moiety is located(directly or indirectly) at the carboxyl (COOH or C) terminus of thesignal sequence. In the chimeric polypeptides of formula (II), thecarboxy (COOH or C) terminus of the amino acid tether is located(directly or indirectly) at the amino (NH₂ or N) terminus of theheterologous starch or dextrin hydrolase moiety. In the chimericpolypeptides of formula (II), the carboxy (COOH or C) terminus of signalsequence is located (directly or indirectly) at the amino (NH₂ or N)terminus of the amino acid tether. Embodiments of chimeric tetheredheterologous polypeptides have been disclosed in WO2018/167670 and areincluded herein in their entirety.

The heterologous nucleic acid molecule can include a thirdpolynucleotide including a promoter capable of controlling theexpression of the first and second polynucleotide. In such embodiment,the promoter and the polynucleotide coding for the signal sequence andthe heterologous polypeptide are operatively linked to one another. Inthe context of the present disclosure, the expressions “operativelylinked” or “operatively associated” refers to fact that the promoter isphysically associated to the first and second polynucleotide in a mannerthat allows, under certain conditions, for expression of theheterologous polypeptide from the heterologous nucleic acid molecule. Inan embodiment, the promoter can be located upstream (5′) of the nucleicacid sequence coding for the heterologous polypeptide. In still anotherembodiment, the promoter can be located downstream (3′) of the nucleicacid sequence coding for the heterologous polypeptide. In the context ofthe present disclosure, one or more than one promoter can be included inthe nucleic acid molecule. When more than one promoter is included inthe nucleic acid molecule, each of the promoters is operatively linkedto the nucleic acid sequence coding for the polypeptide. The promoterscan be located, in view of the nucleic acid molecule coding for thepolypeptide, upstream, downstream as well as both upstream anddownstream.

“Promoter” refers to a DNA fragment capable of controlling theexpression of a coding sequence or functional RNA. The term“expression,” as used herein, refers to the transcription and stableaccumulation of sense (mRNA) from the heterologous nucleic acid moleculedescribed herein. Expression may also refer to translation of mRNA intoa polypeptide. Promoters may be derived in their entirety from a nativegene, or be composed of different elements derived from differentpromoters found in nature, or even comprise synthetic DNA segments. Itis understood by those skilled in the art that different promoters maydirect the expression at different stages of development, or in responseto different environmental or physiological conditions. Promoters whichcause a gene to be expressed in most cells at most times at asubstantial similar level are commonly referred to as “constitutivepromoters”. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical promoter activity. Apromoter is generally bounded at its 3′ terminus by the transcriptioninitiation site and extends upstream (5′ direction) to include theminimum number of bases or elements necessary to initiate transcriptionat levels detectable above background. Within the promoter will be founda transcription initiation site (conveniently defined for example, bymapping with nuclease S1), as well as polypeptide binding domains(consensus sequences) responsible for the binding of the polymerase.

The promoter can be heterologous to the nucleic acid molecule encodingthe heterologous polypeptide. The promoter can be heterologous orderived from a strain being from the same genus or species as therecombinant host cell. In an embodiment, the promoter is derived fromthe same genus or species of the yeast host cell and the polypeptide isderived from different genera that the host cell. One or more promoterscan be used to allow the expression of the polypeptides in therecombinant yeast host cell.

In some embodiments, the recombinant yeast host cell is a facultativeanaerobe, such as Saccharomyces cerevisiae. For facultative anaerobes,cells tend to propagate or ferment depending on the availability ofoxygen. In a fermentation process, yeast cells are generally allowed topropagate before fermentation is conducted. In some embodiments, thepromoter preferentially initiates transcription during a propagationphase such that the heterologous polypeptides (variants or fragments)are expressed during the propagation phase. As used in the context ofthe present disclosure, the expression “propagation phase” refers to anexpansion phase of a commercial process in which the yeasts arepropagated under aerobic conditions. In some instances, the propagatedbiomass can be used in a following fermenting step (e.g. under anaerobicconditions) to maximize the production of one or more desiredmetabolites or fermentation products.

The heterologous nucleic acid molecule can include a promoter (or acombination of promoters) capable of allowing the expression of theheterologous polypeptide during propagation (and optionally duringfermentation). This embodiment will allow the accumulation of thepolypeptide associated with the recombinant yeast host cell prior to anysubsequent use, for example in liquefaction or fermentation. In someembodiments, the promoter substantially limits the expression of thepolypeptide during the propagation phase.

The expression of the polypeptides during the propagation phase mayprovide sufficient expression such that the polypeptide or therecombinant yeast cells may be added during the liquefaction of starch,thereby providing yeast cells with sufficient nutrients to undergometabolic processing. The promoters can be native or heterologous to theheterologous gene encoding the heterologous polypeptide. The promotersthat can be included in the heterologous nucleic acid molecule can beconstitutive or inducible promoters. Constitutive promoters include, butare not limited to, tef2p (e.g., the promoter of the tef2 gene, avariant thereof or a fragment thereof), cwp2p (e.g., the promoter of thecwp2 gene, a variant thereof or a fragment thereof), ssa1p (e.g., thepromoter of the ssa1 gene, a variant thereof), eno1p (e.g., the promoterof the eno1 gene, variant thereof or a fragment thereof), hxk1 (e.g.,the promoter of the hxk1 gene, a variant thereof or a fragment thereof)and/or pgk1p (e.g., the promoter of the pgk1 gene, a variant thereof ora fragment thereof). Inducible promoters include, but are not limited toglucose-regulated promoters (e.g., the promoter of the hxt7 gene(referred to as hxt7p), a functional variant or a functional fragmentthereof; the promoter of the ctt1 gene (referred to as ctt1p), afunctional variant or a functional fragment thereof; the promoter of theglo1 gene (referred to as glo1p), a functional variant or a functionalfragment thereof; the promoter of the ygp1 gene (referred to as ygp1p),a functional variant or a functional fragment thereof; the promoter ofthe gsy2 gene (referred to as gsy2p), a functional variant or afunctional fragment thereof), molasses-regulated promoters (e.g., thepromoter of the mol1 gene (referred to as mol1p), a functional variantor a functional fragment thereof), heat shock-regulated promoters (e.g.,the promoter of the glo1 gene (referred to as glo1p), a functionalvariant or a functional fragment thereof; the promoter of the sti1 gene(referred to as sti1p), a functional variant or a functional fragmentthereof; the promoter of the ygp1 gene (referred to as ygp1p), afunctional variant or a functional fragment thereof; the promoter of thegsy2 gene (referred to as gsy2p), a functional variant or a functionalfragment thereof), oxidative stress response promoters (e.g., thepromoter of the cup1 gene (referred to as cup1p), a functional variantor a functional fragment thereof; the promoter of the ctt1 gene(referred to as ctt1p), a functional variant or a functional fragmentthereof; the promoter of the trx2 gene (referred to as trx2p), afunctional variant or a functional fragment thereof; the promoter of thegpd1 gene (referred to as gpd1p), a functional variant or a functionalfragment thereof; the promoter of the hsp12 gene (referred to ashsp12p), a functional variant or a functional fragment thereof), osmoticstress response promoters (e.g., the promoter of the ctt1 gene (referredto as ctt1p), a functional variant or a functional fragment thereof; thepromoter of the glo1 gene (referred to as glo1p), a functional variantor a functional fragment thereof; the promoter of the gpd1 gene(referred to as gpd1p), a functional variant or a functional fragmentthereof; the promoter of the ygp1 gene (referred to as ygp1p), afunctional variant or a functional fragment thereof), nitrogen-regulatedpromoters (e.g., the promoter of the ygp1 gene (referred to as ygp1p), afunctional variant or a functional fragment thereof) and the promoter ofthe adh1 gene (referred to as adh1p), a functional variant or afunctional fragment thereof) and/or a molasses-regulated promoter (e.g.,the promoter of the tir1 gene (referred to as tir1p), a functionalvariant or a functional fragment thereof).

Promoters that can be included in the heterologous nucleic acid moleculeof the present disclosure include, without limitation, the promoter ofthe tdh1 gene (referred to as tdh1p, a functional variant or afunctional fragment thereof), of the hor7 gene (referred to as hor7p, afunctional variant or a functional fragment thereof), of the hsp150 gene(referred to as hsp150p, a functional variant or a functional fragmentthereof), of the hxt7 gene (referred to as hxt7p, a functional variantor a functional fragment thereof), of the gpm1 gene (referred to asgpm1p, a functional variant or a functional fragment thereof), of thepgk1 gene (referred to as pgk1p, a functional variant or a functionalfragment thereof), of the stl1 gene (referred to as stl1p, a functionalvariant or a functional fragment thereof) and/or of the tef2 gen(referred to as tef2p, a functional variant or a functional fragmentthereof). In an embodiment, the promoter is or comprises the tef2p. Instill another embodiment, the promoter comprises or consists essentiallyof the tdh1p and the hor7p. In a further embodiment, the promoter is thethd1p. In another embodiment, the promoter is the adh1p.

In the context of the present disclosure, the expression “functionalfragment of a promoter” when used in combination to a promoter refers toa shorter nucleic acid sequence than the native promoter which retainthe ability to control the expression of the nucleic acid sequenceencoding the polypeptides during the propagation phase of therecombinant yeast host cells. Usually, functional fragments are either5′ and/or 3′ truncation of one or more nucleic acid residue from thenative promoter nucleic acid sequence.

In some embodiments, the heterologous nucleic acid molecules include oneor a combination of terminator sequence(s) to end the translation of theheterologous polypeptide (or of the chimeric polypeptide comprisingsame). The terminator can be native or heterologous to the nucleic acidsequence encoding the heterologous polypeptide or its correspondingchimera. In some embodiments, one or more terminators can be used. Insome embodiments, the terminator comprises the terminator derived fromis from the dit1 gene (dit1t, a functional variant or a functionalfragment thereof), from the idp1 gene (idp1t, a functional variant or afunctional fragment thereof), from the gpm1 gene (gpm1t, a functionalvariant or a functional fragment thereof), from the pma1 gene (pam1t, afunctional variant or a functional fragment thereof), from the tdh3 gene(tdh3t, a functional variant or a functional fragment thereof), from thehxt2 gene (a functional variant or a functional fragment thereof), fromthe adh3 gene (adh3t, a functional variant or a functional fragmentthereof), and/or from the ira2 gene (ira2t, a functional variant or afunctional fragment thereof). In an embodiment, the terminator comprisesor is derived from the dit1 gene (dit1t, a functional variant or afunctional fragment thereof). In another embodiment, the terminatorcomprises or is derived adh3t and/or idp1t. In the context of thepresent disclosure, the expression “functional variant of a terminator”refers to a nucleic acid sequence that has been substituted in at leastone nucleic acid position when compared to the native terminator whichretain the ability to end the expression of the nucleic acid sequencecoding for the heterologous polypeptide or its corresponding chimera. Inthe context of the present disclosure, the expression “functionalfragment of a terminator” refers to a shorter nucleic acid sequence thanthe native terminator which retain the ability to end the expression ofthe nucleic acid sequence coding for the heterologous polypeptide or itscorresponding chimera.

In some embodiments, the recombinant host cell comprises a geneticmodification (e.g., one or more heterologous nucleic acid molecule)allowing the recombinant expression of the polypeptide having starch ordextrin hydrolase activity. In such embodiment, a heterologous nucleicacid molecule encoding the polypeptide having starch or dextrinhydrolase activity can be introduced in the recombinant host to expressthe polypeptide having starch or dextrin hydrolase activity. Theexpression of the polypeptide having starch or dextrin hydrolaseactivity can be constitutive or induced.

In some embodiments, the recombinant host cell comprises a furthergenetic modification (e.g., the introduction of one or more heterologousnucleic acid molecule) allowing the recombinant expression of thepolypeptide having starch digesting alpha-amylase activity. In suchembodiment, a heterologous nucleic acid molecule encoding thepolypeptide having starch digesting alpha-amylase activity can beintroduced in the recombinant host to express the polypeptide havingstarch digesting alpha-amylase activity activity. The expression of thepolypeptide having starch digesting alpha-amylase activity can beconstitutive or induced. For example, the heterologous alpha-amylase canbe from a Rhizomucor sp., such as, for example, from Rhizomucorpusillus. In an embodiment, the heterologous alpha-amylase correspondsto Uniprot M9T189. In another embodiment, the heterologous alpha-amylasecan have the amino acid sequence of SEQ ID NO: 17, be a variant of theamino acid sequence of SEQ ID NO: 17 having alpha-amylase activity or bea fragment of the amino acid sequence of SEQ ID NO: 17 havingalpha-amylase activity (which can, in an embodiment, correspond to afragment of the amino acid sequence of SEQ ID NO: 17 lacking its signalsequence, e.g., for example, between residues 22 and 471 of SEQ ID NO:17). For example, the heterologous alpha-amylase can be from aAspergillus sp., such as, for example, from Aspergillus luchuensis. Inan embodiment, the heterologous alpha-amylase corresponds to UniprotA0A146F6W4 or to GenBank Accession Number GAT21778. In anotherembodiment, the heterologous alpha-amylase can have the amino acidsequence of SEQ ID NO: 18, be a variant of the amino acid sequence ofSEQ ID NO: 18 having alpha-amylase activity or be a fragment of theamino acid sequence of SEQ ID NO: 18 having alpha-amylase activity(which can, in an embodiment, correspond to a fragment of the amino acidsequence of SEQ ID NO: 18 lacking its signal sequence, e.g., forexample, between residues 22 to 615 of SEQ ID NO: 18). In an embodiment,the heterologous alpha-amylase corresponds to Uniprot 013296 or toGenBank Accession Number BAA22993. In another embodiment, theheterologous alpha-amylase can have the amino acid sequence of SEQ IDNO: 26, be a variant of the amino acid sequence of SEQ ID NO: 26 havingalpha-amylase activity or be a fragment of the amino acid sequence ofSEQ ID NO: 26 having alpha-amylase activity (which can, in anembodiment, correspond to a fragment of the amino acid sequence of SEQID NO: 26 lacking its signal sequence, e.g., for example betweenresidues 22 to 640 of SEQ ID NO: 26). For example, the heterologousalpha-amylase can be from Aspergillus oryzae. In an embodiment, theheterologous alpha-amylase corresponds to Uniprot Q2UIS5 or to GenBankAccession Number XP_001820542. In another embodiment, the heterologousalpha-amylase can have the amino acid sequence of SEQ ID NO: 19, be avariant of the amino acid sequence of SEQ ID NO: 19 having alpha-amylaseactivity or be a fragment of the amino acid sequence of SEQ ID NO: 19having alpha-amylase activity (which can, in an embodiment, correspondto a fragment of the amino acid sequence of SEQ ID NO: 19 lacking itssignal sequence, e.g., for example between residues 24 to 549 of SEQ IDNO: 19). For example, the heterologous alpha-amylase can be fromAspergillus niger. In an embodiment, the heterologous alpha-amylasecorresponds to Uniprot A2QTS4 or to GenBank Accession NumberXP_001393626. In another embodiment, the heterologous alpha-amylase canhave the amino acid sequence of SEQ ID NO: 21, be a variant of the aminoacid sequence of SEQ ID NO: 21 having alpha-amylase activity or be afragment of the amino acid sequence of SEQ ID NO: 21 havingalpha-amylase activity (which can, in an embodiment, correspond to afragment of the amino acid sequence of SEQ ID NO: 21 lacking its signalsequence, e.g., for example between residues 26 to 555 of SEQ ID NO:21). In an embodiment, the heterologous alpha-amylase corresponds toUniprot A2R6F9 or to GenBank Accession Number XP_001397301. In anotherembodiment, the heterologous alpha-amylase can have the amino acidsequence of SEQ ID NO: 22, be a variant of the amino acid sequence ofSEQ ID NO: 22 having alpha-amylase activity or be a fragment of theamino acid sequence of SEQ ID NO: 22 having alpha-amylase activity(which can, in an embodiment, correspond to a fragment of the amino acidsequence of SEQ ID NO: 22 lacking its signal sequence, e.g., for examplebetween residues 23 and 567 of SEQ ID NO: 22). In an embodiment, theheterologous alpha-amylase corresponds to GenBank Accession NumberXP_001395328. In another embodiment, the heterologous alpha-amylase canhave the amino acid sequence of SEQ ID NO: 23, be a variant of the aminoacid sequence of SEQ ID NO: 23 having alpha-amylase activity or be afragment of the amino acid sequence of SEQ ID NO: 23 havingalpha-amylase activity (which can, in an embodiment, correspond to afragment of the amino acid sequence of SEQ ID NO: 23 lacking its signalsequence, e.g., for example between residues 30 and 550 of SEQ ID NO:23). In an embodiment, the heterologous alpha-amylase corresponds toUniprot A0A370BQ30 or to GenBank Accession Number RDH15462. In anotherembodiment, the heterologous alpha-amylase can have the amino acidsequence of SEQ ID NO: 24, be a variant of the amino acid sequence ofSEQ ID NO: 24 having alpha-amylase activity or be a fragment of theamino acid sequence of SEQ ID NO: 24 having alpha-amylase activity(which can, in an embodiment, correspond to a fragment of the amino acidsequence of SEQ ID NO: 24 lacking its signal sequence, e.g., for examplebetween residues 17 and 524 of SEQ ID NO: 24). For example, theheterologous alpha-amylase can be from Aspergillus fischeri. In anembodiment, the heterologous alpha-amylase corresponds to Uniprot A1CYB1or to GenBank Accession Number XP_001265628. In another embodiment, theheterologous alpha-amylase can have the amino acid sequence of SEQ IDNO: 25, be a variant of the amino acid sequence of SEQ ID NO: 25 havingalpha-amylase activity or be a fragment of the amino acid sequence ofSEQ ID NO: 25 having alpha-amylase activity (which can, in anembodiment, correspond to a fragment of the amino acid sequence of SEQID NO: 25 lacking its signal sequence, e.g., for example betweenresidues 24 to 632 of SEQ ID NO: 25). For example, the heterologousalpha-amylase can be from a Homo sp., such as, for example, from Homosapiens. In an embodiment, the heterologous alpha-amylase corresponds toGenBank Accession Number 1B2Y_A. In another embodiment, the heterologousalpha-amylase can have the amino acid sequence of SEQ ID NO: 20, be avariant of the amino acid sequence of SEQ ID NO: 20 having alpha-amylaseactivity or be a fragment of the amino acid sequence of SEQ ID NO: 20having alpha-amylase activity (which can, in an embodiment, correspondto a fragment of the amino acid sequence of SEQ ID NO: 20 lacking itssignal sequence, e.g., for example between residues 20 to 515 of SEQ IDNO: 20).

A variant alpha-amylase comprises at least one amino acid difference(substitution or addition) when compared to the amino acid sequence ofthe alpha-amylase polypeptide of SEQ ID NO: 17 to 26 and still exhibitsalpha-amylase activity. In an embodiment, the variant alpha-amylaseexhibits at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the activity of thenative or wild-type alpha-amylase having the amino acid sequence of SEQID NO: 17 to 26. The alpha-amylase variants also have at least 70%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity when compared to the wild-type ornative alpha-amylase having the amino acid sequence of SEQ ID NO: 17 to26 over its entire length. The term “percent identity”, as known in theart, is a relationship between two or more polypeptide sequences, asdetermined by comparing the sequences. The level of identity can bedetermined conventionally using known computer programs. Identity can bereadily calculated by known methods, including but not limited to thosedescribed in: Computational Molecular Biology (Lesk, A. M., ed.) OxfordUniversity Press, N Y (1988); Biocomputing: Informatics and GenomeProjects (Smith, D. W., ed.) Academic Press, N Y (1993); ComputerAnalysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G.,eds.) Humana Press, N J (1994); Sequence Analysis in Molecular Biology(von Heinje, G., ed.) Academic Press (1987); and Sequence AnalysisPrimer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991).Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignments of thesequences disclosed herein were performed using the Clustal method ofalignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLB 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The variant alpha-amylases described herein may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide for purification of the polypeptide.Conservative substitutions typically include the substitution of oneamino acid for another with similar characteristics, e.g., substitutionswithin the following groups: valine, glycine; glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. Other conservative amino acid substitutions are known in theart and are included herein. Non-conservative substitutions, such asreplacing a basic amino acid with a hydrophobic one, are also well-knownin the art.

A variant alpha-amylase can also be a conservative variant or an allelicvariant. As used herein, a conservative variant refers to alterations inthe amino acid sequence that do not adversely affect the biologicalfunctions of the starch digesting alpha-amylase. A substitution,insertion or deletion is said to adversely affect the polypeptide whenthe altered sequence prevents or disrupts a biological functionassociated with the starch digesting alpha-amylase (e.g., the hydrolysisof starch into glucose). For example, the overall charge, structure orhydrophobic-hydrophilic properties of the polypeptide can be alteredwithout adversely affecting a biological activity. Accordingly, theamino acid sequence can be altered, for example to render the peptidemore hydrophobic or hydrophilic, without adversely affecting thebiological activities of the starch digesting alpha-amylase.

The present disclosure also provide fragments of the alpha-amylases andalpha-amylases variants described herein. A fragment comprises at leastone less amino acid residue when compared to the amino acid sequence ofthe catalytic domain or the alpha-amylase polypeptide or variant andstill possess the enzymatic activity of the full-length alpha-amylase.In an embodiment, the alpha-amylase fragment exhibits at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% activity when compared to the full-lengthalpha-amylase having the amino acid of SEQ ID NO: 17 to 26 or variantsthereof. The alpha-amylase fragments can also have at least 70%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity when compared to the alpha-amylasehaving the amino acid sequence of SEQ ID NO: 17 to 26 or variantsthereof. The fragment can be, for example, a truncation of one or moreamino acid residues at the amino-terminus, the carboxy terminus or bothtermini of the starch digesting alpha-amylase or variant. In a specificembodiment, the fragment corresponds to a polypeptide of any one of SEQID NO: 17 to 26 to which the signal sequence has been removed.Alternatively or in combination, the fragment can be generated fromremoving one or more internal amino acid residues. In an embodiment, thealpha-amylase fragment has at least 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600 or more consecutive amino acids of the alpha-amylasehaving the amino acid sequence of SEQ ID NO: 17 to 26 or variantsthereof.

The alpha-amylase can be expressed using its native signal sequence orcan be expressed using a heterologous signal sequence. In embodimentsthe heterologous signal sequence associated with the alpha-amylase canhave the amino acid sequence of SEQ ID NO: 5 or the section spanningresidues 1 to 21 of SEQ ID NO: 17, 1 to 21 of SEQ ID NO: 18, 1 to 23 ofSEQ ID NO: 19, 1 to 19 of SEQ ID NO: 20, 1 to 25 of SEQ ID NO: 21, 1 to22 of SEQ ID NO: 22, 1 to 29 of SEQ ID NO: 23, 1 to 16 of SEQ ID NO: 24,1 to 23 of SEQ ID NO: 25, 1 to 21 of SEQ ID NO: 26, 1 to 17 of SEQ IDNO: 27, 1 to 20 of SEQ ID NO: 28, 1 to 22 of SEQ ID NO: 29, 1 to 18 ofSEQ ID NO: 30, 1 to 25 of SEQ ID NO: 31, 1 to 19 of SEQ ID NO: 32, 1 to18 of SEQ ID NO: 33, 1 to 19 of SEQ ID NO: 34, 1 to 18 of SEQ ID NO: 35,1 to 18 of SEQ ID NO: 36 as well as variants and fragments thereof.

Process for Saccharification and Fermentation of a Biomass

The recombinant yeast host cells described herein can be used insaccharification for improving the hydrolysis of a biomass and, in someembodiments, the production of a fermentation product from the biomass.In some embodiments, the recombinant yeast host cells of the presentdisclosure maintain their robustness during saccharification andfermentation in the presence of a stressor such as, for example, lacticacid, formic acid and/or a bacterial contamination (that can beassociated, in some embodiments, with an increase in lactic acid duringfermentation), a decrease in pH, a reduction in aeration, elevatedtemperatures or a combination of these conditions.

The fermented product intended to be obtained during the fermentationcan be an alcohol, such as, for example, ethanol, isopropanol,n-propanol, 1-butanol, methanol, acetone, 1,3-propanediol and/or1,2-propanediol. In an embodiment, the fermented product is ethanol.

In the process, the biomass that can be hydrolyzed (and optionallyfermented) with the recombinant yeast host cells. Such biomass includesany type of biomass known in the art and described herein. For example,the biomass can include, but is not limited to, starch, sugar andlignocellulosic materials. Starch materials can include, but are notlimited to, mashes such as corn, wheat, rye, barley, rice, or milo.Sugar materials can include, but are not limited to, sugar beets,artichoke tubers, sweet sorghum, molasses or cane. The terms“lignocellulosic material”, “lignocellulosic substrate” and “cellulosicbiomass” mean any type of substrate comprising cellulose, hemicellulose,lignin, or combinations thereof, such as but not limited to woodybiomass, forage grasses, herbaceous energy crops, non-woody-plantbiomass, agricultural wastes and/or agricultural residues, forestryresidues and/or forestry wastes, paper-production sludge and/or wastepaper sludge, waste-water-treatment sludge, municipal solid waste, cornfiber from wet and dry mill corn ethanol plants and sugar-processingresidues. The terms “hemicellulosics”, “hemicellulosic portions” and“hemicellulosic fractions” mean the non-lignin, non-cellulose elementsof lignocellulosic material, such as but not limited to hemicellulose(i.e., comprising xyloglucan, xylan, glucuronoxylan, arabinoxylan,mannan, glucomannan and galactoglucomannan), pectins (e.g.,homogalacturonans, rhamnogalacturonan I and II, and xylogalacturonan)and proteoglycans (e.g., arabinogalactan-polypeptide, extensin, and proline-rich polypeptides).

In a non-limiting example, the lignocellulosic material can include, butis not limited to, woody biomass, such as recycled wood pulp fiber,sawdust, hardwood, softwood, and combinations thereof; grasses, such asswitch grass, cord grass, rye grass, reed canary grass, miscanthus, or acombination thereof; sugar-processing residues, such as but not limitedto sugar cane bagasse; agricultural wastes, such as but not limited torice straw, rice hulls, barley straw, corn cobs, cereal straw, wheatstraw, canola straw, oat straw, oat hulls, and corn fiber; stover, suchas but not limited to soybean stover, corn stover; succulents, such asbut not limited to, agave; and forestry wastes, such as but not limitedto, recycled wood pulp fiber, sawdust, hardwood (e.g., poplar, oak,maple, birch, willow), softwood, or any combination thereof.Lignocellulosic material may comprise one species of fiber;alternatively, lignocellulosic material may comprise a mixture of fibersthat originate from different lignocellulosic materials. Otherlignocellulosic materials are agricultural wastes, such as cerealstraws, including wheat straw, barley straw, canola straw and oat straw;corn fiber; stovers, such as corn stover and soybean stover; grasses,such as switch grass, reed canary grass, cord grass, and miscanthus; orcombinations thereof.

Substrates for cellulose activity assays can be divided into twocategories, soluble and insoluble, based on their solubility in water.Soluble substrates include cellodextrins or derivatives, carboxymethylcellulose (CMC), or hydroxyethyl cellulose (HEC). Insoluble substratesinclude crystalline cellulose, microcrystalline cellulose (Avicel),amorphous cellulose, such as phosphoric acid swollen cellulose (PASO),dyed or fluorescent cellulose, and pretreated lignocellulosic biomass.These substrates are generally highly ordered cellulosic material andthus only sparingly soluble.

It will be appreciated that suitable lignocellulosic material may be anyfeedstock that contains soluble and/or insoluble cellulose, where theinsoluble cellulose may be in a crystalline or non-crystalline form. Invarious embodiments, the lignocellulosic biomass comprises, for example,wood, corn, corn stover, sawdust, bark, molasses, sugarcane, leaves,agricultural and forestry residues, grasses such as switchgrass,ruminant digestion products, municipal wastes, paper mill effluent,newspaper, cardboard or combinations thereof.

Paper sludge is also a viable feedstock for lactate or acetateproduction. Paper sludge is solid residue arising from pulping andpaper-making, and is typically removed from process wastewater in aprimary clarifier. The cost of disposing of wet sludge is a significantincentive to convert the material for other uses, such as conversion toethanol. Processes provided by the present invention are widelyapplicable. Moreover, the hydrolyzed biomass may be used to produceethanol or higher value added chemicals, such as organic acids,aromatics, esters, acetone and polymer intermediates.

The process of the present disclosure comprise contacting therecombinant host cells comprising the heterologous polypeptide havingstarch or dextrin hydrolase activity described herein with a biomass soas to allow the hydrolysis of at least a part of the biomass and theconversion of the biomass (at least in part) into a fermentation product(e.g., an alcohol such as ethanol). In some embodiments, the biomass tobe hydrolyzed/fermented is a lignocellulosic biomass and, in someembodiments, it comprises starch (in a gelatinized or raw form). In anembodiment, the biomass to be hydrolyzed/fermented is raw starch. Inother embodiments, the biomass to be hydrolyzed/fermented is derivedfrom corn, potato, cassava, rice, or buckwheat. In preferredembodiments, the biomass is derived from corn, such as in the form ofcorn mash. The process can include, in some embodiments, heating thelignocellulosic biomass prior to fermentation to provide starch in agelatinized form. In another embodiment, the biomass comprises or isderived from sugar cane.

The fermentation process can be performed at temperatures of at leastabout 20° C., about 21° C., about 22° C., about 23° C., about 24° C.,about 25° C., about 26° C., about 27° C., about 28° C., about 29° C.,about 30° C., about 31° C., about 32° C., about 33°, about 34° C., about35° C., about 36° C., about 37° C., about 38° C., about 39° C., about40° C., about 41° C., about 42° C., about 43° C., about 44° C., about45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about50° C. In some embodiments, the production of ethanol from cellulose canbe performed, for example, at temperatures above about 30° C., about 31°C., about 32° C., about 33° C., about 34° C., about 35° C., about 36°C., about 37° C., about 38° C., about 39° C., about 40° C., about 41°C., about 42° C., or about 43° C., or about 44° C., or about 45° C., orabout 50° C. In some embodiments, the recombinant microbial host cellcan produce ethanol from cellulose at temperatures from about 30° C. to60° C., about 30° C. to 55° C., about 30° C. to 50° C., about 40° C. to60° C., about 40° C. to 55° C. or about 40° C. to 50° C.

In some embodiments, the liquefaction of starch occurs in the presenceof recombinant yeast host cells described herein. In some embodiments,the liquefaction of starch is maintained at a temperature of betweenabout 70° C.-105° C. to allow for proper gelatinization and hydrolysisof the starch. In an embodiment, the liquefaction occurs at atemperature of at least about 70° C., 75° C., 80° C., 85° C., 90° C.,95° C., 100° C. or 105° C. Alternatively or in combination, theliquefaction occurs at a temperate of no more than about 105° C., 100°C., 95° C., 90° C., 85° C., 80° C., 75° C. or 70° C. In yet anotherembodiment, the liquefaction occurs at a temperature between about 80°C. and 85° C. (which can include a thermal treatment spike at 105° C.).

In some embodiments, the process can be used to produce ethanol at aparticular rate. For example, in some embodiments, ethanol is producedat a rate of at least about 0.1 mg per hour per liter, at least about0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, atleast about 0.75 mg per hour per liter, at least about 1.0 mg per hourper liter, at least about 2.0 mg per hour per liter, at least about 5.0mg per hour per liter, at least about 10 mg per hour per liter, at leastabout 15 mg per hour per liter, at least about 20.0 mg per hour perliter, at least about 25 mg per hour per liter, at least about 30 mg perhour per liter, at least about 50 mg per hour per liter, at least about100 mg per hour per liter, at least about 200 mg per hour per liter, atleast about 300 mg per hour per liter, at least about 400 mg per hourper liter, at least about 500 mg per hour per liter, at least about 600mg per hour per liter, at least about 700 mg per hour per liter, atleast about 800 mg per hour per liter, at least about 900 mg per hourper liter, at least about 1 g per hour per liter, at least about 1.5 gper hour per liter, at least about 2 g per hour per liter, at leastabout 2.5 g per hour per liter, at least about 3 g per hour per liter,at least about 3.5 g per hour per liter, at least about 4 g per hour perliter, at least about 4.5 g per hour per liter, at least about 5 g perhour per liter, at least about 5.5 g per hour per liter, at least about6 g per hour per liter, at least about 6.5 g per hour per liter, atleast about 7 g per hour per liter, at least about 7.5 g per hour perliter, at least about 8 g per hour per liter, at least about 8.5 g perhour per liter, at least about 9 g per hour per liter, at least about9.5 g per hour per liter, at least about 10 g per hour per liter, atleast about 10.5 g per hour per liter, at least about 11 g per hour perliter, at least about 11.5 g per hour per liter, at least about 12 g perhour per liter, at least about 12.5 g per hour per liter, at least about13 g per hour per liter, at least about 13.5 g per hour per liter, atleast about 14 g per hour per liter, at least about 14.5 g per hour perliter or at least about 15 g per hour per liter.

Ethanol yield per fermentation batch can be measured using one of atleast four methods: 1) measurement of total gallons of ethanol distilledper bushel of corn ground for the processing of each fermentation batch;2) measurement of ethanol concentration (titer) in the fermentation beersoluble fraction (supernatant) at the end of fermentation before thebeer is pumped to the beer well, normalized to the average total solids(total dry matter) concentration of the liquefact used to fill thefermentation batch (“ethanol yield per liquefact solids”); 3)measurement of ethanol concentration in the fermentation beer solublefraction (supernatant) at the end of fermentation before the beer ispumped to the beer well, normalized to the average total solids (totaldry matter) concentration of the slurry used to produce the liquefactfilling the fermentation batch (“ethanol yield per slurry solids”); and4) measurement of ethanol concentration in the fermentation beer solublefraction (supernatant) at the end of fermentation before the beer ispumped to the beer well, normalized to the average total solids (totaldry matter) concentration of the liquefact mash filling the fermentationbatch, with the liquefact mash total solids (total dry matter) adjustedfor the recycled unfermented total solids (total dry matter) in therecycled thin stillage (backset): “ethanol yield per fermentable mashsolids.” It is possible to track total ethanol production and cornconsumption on a daily basis, rather than on a per batch basis, and sothe second, third, and fourth ethanol yield measurement methods perfermentation batch are most commonly used to assess batch ethanol yield.Where changes in the concentration of unfermented total solids in therecycled thin stillage or changes in the relative volumetric flow rateof the recycled thin stillage to the total liquefact flow rate mayoccur, the fourth method of measuring ethanol yield per fermentationbatch is preferable (ethanol yield per fermentable mash solids). Forproduction facilities that do not frequently measure total solids (totaldry matter) of recycled thin stillage, or that do not track the relativevolumetric flow rate of recycled thin stillage to that of the totalliquefact on a per batch basis, the second or third ethanol yieldmeasurement methods are typically applied.

Samples of fermentation beer can be taken periodically (often every 6 h,10 h, or 12 h) over the course of each fermentation batch to evaluatethe progress of the fermentation batch as glucose is converted toethanol, and then at the end of the fermentation batch (at fermentationdrop) as part of evaluating the final ethanol yield for the batch.Samples can either be centrifuged to remove suspended solids with thesupernatant collected, or allowed to drip through filter paper to removesuspended solids with the filtrate collected. The sample supernatant orfiltrate can be filtered (through a 0.22 μm pore size syringe filtercartridge for example), and the clarified filtrate collected in an HPLCvial. The clarified filtrate is analyzed using high-performance liquidchromatography (HPLC) to measure the concentration of solublefermentation components including: ethanol, glycerol, glucose(dextrose), lactic acid, acetic acid, and glucose di- and trisaccharides(DP2 and DP3). Higher molecular weight glucose oligomers (often referredto as “DP4+”) are typically measured as part of a single aggregate peakthat elutes with the void volume of the HPLC column. The sum of theconcentrations of glucose, DP2, DP3, and DP4+ as measured in the beersoluble fraction by HPLC is referred to as the “total residual sugar”concentration.

Ethanol production can be measured using any method known in the art.For example, the quantity of ethanol in fermentation samples can beassessed using HPLC analysis. Many ethanol assay kits are commerciallyavailable that use, for example, alcohol oxidase enzyme based assays.

In some embodiments, the process can be used in the presence of astressor such as low pH. For example, the stressor is a pH of 7.0 orlower, 6.5 or lower, 6.0 or lower, 5.5 or lower, 5.0 or lower, 4.8 orlower, 4.6 or lower, 4.4 or lower, 4.2 or lower, 4.0 or lower, 3.8 orlower, 3.6 or lower, 3.4 or lower, 3.2 or lower, or 3.0 or lower.

As shown in the Examples, recombinant yeast host cells expressing theheterologous glucoamylase exhibits enhanced robustness compared to yeasthost cells expressing other known glucoamylases. In specific embodimentsof a recombinant yeast host cell expressing a heterologous glucoamylase,fermentation with the recombinant yeast cell yielded higher ethanoltiters than recombinant yeast host cells expressing other heterologousglucoamylases. In some embodiments, the recombinant yeast host cellexpressing heterologous glucoamylase yielded greater than 10 mg/Lincrease, greater than 25 mg/L increase, greater than 50 mg/L increase,greater than 100 mg/L increase, greater than 200 mg/L increase, greaterthan 300 mg/L increase, greater than 400 mg/L increase, greater than 500mg/L increase, greater than 600 mg/L increase, greater than 700 mg/Lincrease, greater than 800 mg/L increase, greater than 900 mg/Lincrease, or greater than 1 g/L increase) in ethanol production at lowpH values and in corn fermentation when compared to recombinant yeasthost cells expressing other heterologous glucoamylases.

In the process described herein, it is possible to add an exogenoussource (e.g., to dose) of an enzyme to facilitate saccharification orimprove fermentation yield. As such, the process can comprise includingone or more dose(s) of one or more enzyme(s) during thesaccharificaction and/or the fermentation step. The exogenous enzymethat can be used during the saccharification/fermentation process caninclude, without limitation, an alpha-amylase, a glucoamylase, aprotease, a phytase, a pullulanase, a cellulase, a hemi-cellulase suchas a xylanase, a trehalase, or any combination thereof. The enzyme canbe purified and/or provided as part of a cocktail. In the process of thepresent disclosure, a reduced dose or amount of an enzyme can be used incombination with the recombinant yeast host cell of the presentdisclosure to complete the fermentation.

The process of the present disclosure can include a step of adding adose (or multiple doses) of an exogenous enzyme (which may be purified)to increase the fermentation yield or allow the yeast to complete thefermentation. In such embodiment, the requirement to add one or moredose(s) can be determined prior to or during fermentation.

For example, the exogenous glucoamylase can be from a Gloeophyllum sp.,such as, for example, from Gloeophyllum trabeum. In an embodiment, theexogenous glucoamylase corresponds to Uniprot S7Q4V9 or GenBankAccession Number_007866834. In another embodiment, the exogenousglucoamylase can have the amino acid sequence of SEQ ID NO: 27, be avariant of the amino acid sequence of SEQ ID NO: 27 having glucoamylaseactivity or be a fragment of the amino acid sequence of SEQ ID NO: 27having glucoamylase activity (which can, in an embodiment, correspond toa fragment of the amino acid sequence of SEQ ID NO: 27 lacking itssignal sequence). For example, the exogenous glucoamylase can be from aTrichoderma sp., such as, for example, from Trichoderma reesii. In anembodiment, the exogenous glucoamylase corresponds to Uniprot G0R866 orGenBank Accession Number_XP_006960925. In another embodiment, theexogenous glucoamylase can have the amino acid sequence of SEQ ID NO:28, be a variant of the amino acid sequence of SEQ ID NO: 28 havingglucoamylase activity or be a fragment of the amino acid sequence of SEQID NO: 28 having glucoamylase activity (which can, in an embodiment,correspond to a fragment of the amino acid sequence of SEQ ID NO: 28lacking its signal sequence). For example, the exogenous glucoamylasecan be from a Trametes sp., such as, for example, from Trametescingulata. In another embodiment, the exogenous glucoamylase can havethe amino acid sequence of SEQ ID NO: 29, be a variant of the amino acidsequence of SEQ ID NO: 29 having glucoamylase activity or be a fragmentof the amino acid sequence of SEQ ID NO: 29 having glucoamylase activity(which can, in an embodiment, correspond to a fragment of the amino acidsequence of SEQ ID NO: 29 lacking its signal sequence). For example, theexogenous glucoamylase can be from a Athelia sp., such as, for example,from Athelia rolfsil. In an embodiment, the exogenous glucoamylasecorresponds to Uniprot Q12596 or GenBank Accession Number_BAA08436. Inanother embodiment, the exogenous glucoamylase can have the amino acidsequence of SEQ ID NO: 30, be a variant of the amino acid sequence ofSEQ ID NO: 30 having glucoamylase activity or be a fragment of the aminoacid sequence of SEQ ID NO: 30 having glucoamylase activity (which can,in an embodiment, correspond to a fragment of the amino acid sequence ofSEQ ID NO: 30 lacking its signal sequence). For example, the exogenousglucoamylase can be from a Rhizopus sp., such as, for example, fromRhizopus oryzae In an embodiment, the exogenous glucoamylase correspondsto Uniprot P07683 or GenBank Accession Number P07683. In anotherembodiment, the exogenous glucoamylase can have the amino acid sequenceof SEQ ID NO: 31, be a variant of the amino acid sequence of SEQ ID NO:31 having glucoamylase activity or be a fragment of the amino acidsequence of SEQ ID NO: 31 having glucoamylase activity (which can, in anembodiment, correspond to a fragment of the amino acid sequence of SEQID NO: 31 lacking its signal sequence). For example, the exogenousglucoamylase can be from a Aspergillus sp., such as, for example, fromAspergillus oryzae. In an embodiment, the exogenous glucoamylasecorresponds to Uniprot P36914 or GenBank Accession Number BAA00841. Inanother embodiment, the exogenous glucoamylase can have the amino acidsequence of SEQ ID NO: 32, be a variant of the amino acid sequence ofSEQ ID NO: 32 having glucoamylase activity or be a fragment of the aminoacid sequence of SEQ ID NO: 32 having glucoamylase activity (which can,in an embodiment, correspond to a fragment of the amino acid sequence ofSEQ ID NO: 32 lacking its signal sequence). In yet another example, theexogenous heterologousglucoamylase can be from Aspergillus awamori. Inan embodiment, the exogenous glucoamylase corresponds to Uniprot Q76L97or GenBank Accession Number BAD06004. In another embodiment, theexogenous glucoamylase can have the amino acid sequence of SEQ ID NO:35, be a variant of the amino acid sequence of SEQ ID NO: 35 havingglucoamylase activity or be a fragment of the amino acid sequence of SEQID NO: 35 having glucoamylase activity (which can, in an embodiment,correspond to a fragment of the amino acid sequence of SEQ ID NO: 35lacking its signal sequence). In yet another example, the exogenousglucoamylase can be from Aspergillus niger. In an embodiment, theexogenous glucoamylase corresponds to Uniprot Q870G8 or GenBankAccession Number AAP04499. In another embodiment, the exogenousglucoamylase can have the amino acid sequence of SEQ ID NO: 36, be avariant of the amino acid sequence of SEQ ID NO: 36 having glucoamylaseactivity or be a fragment of the amino acid sequence of SEQ ID NO: 36having glucoamylase activity (which can, in an embodiment, correspond toa fragment of the amino acid sequence of SEQ ID NO: 36 lacking itssignal sequence). For example, the exogenous glucoamylase can be from aOphiostoma sp., such as, for example, from Ophiostoma floccosum. In anembodiment, the exogenous glucoamylase corresponds to Uniprot Q06SN2 orGenBank Accession Number ABF72529. In another embodiment, the exogenousglucoamylase can have the amino acid sequence of SEQ ID NO: 33, be avariant of the amino acid sequence of SEQ ID NO: 33 having glucoamylaseactivity or be a fragment of the amino acid sequence of SEQ ID NO: 33having glucoamylase activity (which can, in an embodiment, correspond toa fragment of the amino acid sequence of SEQ ID NO: 33 lacking itssignal sequence). For example, the exogenous glucoamylase can be from aTrichocladium sp., such as, for example, from Trichocladium griseum. Inan embodiment, the exogenous glucoamylase corresponds to Uniprot Q12623or GenBank Accession Number AAA33386. In another embodiment, theexogenous glucoamylase can have the amino acid sequence of SEQ ID NO:34, be a variant of the amino acid sequence of SEQ ID NO: 34 havingglucoamylase activity or be a fragment of the amino acid sequence of SEQID NO: 34 having glucoamylase activity (which can, in an embodiment,correspond to a fragment of the amino acid sequence of SEQ ID NO: 34lacking its signal sequence).

For example, the exogenous alpha-amylase can be from a Rhizomucor sp.,such as, for example, from Rhizomucor pusillus. In an embodiment, theexogenous alpha-amylase corresponds to Uniprot M9T189. In anotherembodiment, the exogenous alpha-amylase can have the amino acid sequenceof SEQ ID NO: 17, be a variant of the amino acid sequence of SEQ ID NO:17 having alpha-amylase activity or be a fragment of the amino acidsequence of SEQ ID NO: 17 having alpha-amylase activity (which can, inan embodiment, correspond to a fragment of the amino acid sequence ofSEQ ID NO: 17 lacking its signal sequence). For example, the exogenousalpha-amylase can be from a Aspergillus sp., such as, for example, fromAspergillus luchuensis. In an embodiment, the exogenous alpha-amylasecorresponds to Uniprot A0A146F6W4 or to GenBank Accession NumberGAT21778. In another embodiment, the exogenous alpha-amylase can havethe amino acid sequence of SEQ ID NO: 18, be a variant of the amino acidsequence of SEQ ID NO: 18 having alpha-amylase activity or be a fragmentof the amino acid sequence of SEQ ID NO: 18 having alpha-amylaseactivity (which can, in an embodiment, correspond to a fragment of theamino acid sequence of SEQ ID NO: 18 lacking its signal sequence). In anembodiment, the exogenous alpha-amylase corresponds to Uniprot 013296 orto GenBank Accession Number BAA22993. In another embodiment, theexogenous alpha-amylase can have the amino acid sequence of SEQ ID NO:26, be a variant of the amino acid sequence of SEQ ID NO: 26 havingalpha-amylase activity or be a fragment of the amino acid sequence ofSEQ ID NO: 26 having alpha-amylase activity (which can, in anembodiment, correspond to a fragment of the amino acid sequence of SEQID NO: 26 lacking its signal sequence). For example, the exogenousalpha-amylase can be from Aspergillus oryzae. In an embodiment, theexogenous alpha-amylase corresponds to Uniprot Q2UIS5 or to GenBankAccession Number XP_001820542. In another embodiment, the exogenousalpha-amylase can have the amino acid sequence of SEQ ID NO: 19, be avariant of the amino acid sequence of SEQ ID NO: 19 having alpha-amylaseactivity or be a fragment of the amino acid sequence of SEQ ID NO: 19having alpha-amylase activity (which can, in an embodiment, correspondto a fragment of the amino acid sequence of SEQ ID NO: 19 lacking itssignal sequence). For example, the exogenous alpha-amylase can be fromAspergillus niger. In an embodiment, the exogenous alpha-amylasecorresponds to Uniprot A2QTS4 or to GenBank Accession NumberXP_001393626. In another embodiment, the exogenous alpha-amylase canhave the amino acid sequence of SEQ ID NO: 21, be a variant of the aminoacid sequence of SEQ ID NO: 21 having alpha-amylase activity or be afragment of the amino acid sequence of SEQ ID NO: 21 havingalpha-amylase activity (which can, in an embodiment, correspond to afragment of the amino acid sequence of SEQ ID NO: 21 lacking its signalsequence). In an embodiment, the exogenous alpha-amylase corresponds toUniprot A2R6F9 or to GenBank Accession Number XP_001397301. In anotherembodiment, the exogenous alpha-amylase can have the amino acid sequenceof SEQ ID NO: 22, be a variant of the amino acid sequence of SEQ ID NO:22 having alpha-amylase activity or be a fragment of the amino acidsequence of SEQ ID NO: 22 having alpha-amylase activity (which can, inan embodiment, correspond to a fragment of the amino acid sequence ofSEQ ID NO: 22 lacking its signal sequence). In an embodiment, theexogenous alpha-amylase corresponds to GenBank Accession NumberXP_001395328. In another embodiment, the exogenous alpha-amylase canhave the amino acid sequence of SEQ ID NO: 23, be a variant of the aminoacid sequence of SEQ ID NO: 23 having alpha-amylase activity or be afragment of the amino acid sequence of SEQ ID NO: 23 havingalpha-amylase activity (which can, in an embodiment, correspond to afragment of the amino acid sequence of SEQ ID NO: 23 lacking its signalsequence). In an embodiment, the exogenous alpha-amylase corresponds toUniprot A0A370BQ30 or to GenBank Accession Number RDH15462. In anotherembodiment, the exogenous alpha-amylase can have the amino acid sequenceof SEQ ID NO: 24, be a variant of the amino acid sequence of SEQ ID NO:24 having alpha-amylase activity or be a fragment of the amino acidsequence of SEQ ID NO: 24 having alpha-amylase activity (which can, inan embodiment, correspond to a fragment of the amino acid sequence ofSEQ ID NO: 24 lacking its signal sequence). For example, the exogenousalpha-amylase can be from Aspergillus fischeri. In an embodiment, theexogenous alpha-amylase corresponds to Uniprot A1CYB1 or to GenBankAccession Number XP_001265628. In another embodiment, the exogenousalpha-amylase can have the amino acid sequence of SEQ ID NO: 25, be avariant of the amino acid sequence of SEQ ID NO: 25 having alpha-amylaseactivity or be a fragment of the amino acid sequence of SEQ ID NO: 25having alpha-amylase activity (which can, in an embodiment, correspondto a fragment of the amino acid sequence of SEQ ID NO: 25 lacking itssignal sequence). For example, the exogenous alpha-amylase can be from aHomo sp., such as, for example, from Homo sapiens. In an embodiment, theexogenous alpha-amylase corresponds to GenBank Accession Number 1B2Y_A.In another embodiment, the exogenous alpha-amylase can have the aminoacid sequence of SEQ ID NO: 20, be a variant of the amino acid sequenceof SEQ ID NO: 20 having alpha-amylase activity or be a fragment of theamino acid sequence of SEQ ID NO: 20 having alpha-amylase activity(which can, in an embodiment, correspond to a fragment of the amino acidsequence of SEQ ID NO: 20 lacking its signal sequence).

In some embodiments in which the exogenous enzyme is a glucoamylase, itmay be beneficial to not over-dose or under-dose the exogenous enzyme.In early- and mid-fermentation, the starch dextrin hydrolysis reactionproducing glucose catalyzed by glucoamylase (GA) is substrate-rich. As aresult, the hydrolysis reaction rate is primarily controlled by the GAcatalyst concentration and the specific reaction frequency (the enzymeturnover frequency) for the GA enzyme protein at the fermentationtemperature. At the beginning of fermentation, as the fermentor beginsto be filled by liquefact and as either a mash propagation batch or adirect pitch yeast addition are used to inoculate fermentation, theyeast cell concentration in fermentation is at a minimum. Over the first24-30 hours of fermentation the yeast population increases due to cellgrowth and division. If GA enzyme is over-dosed in early fermentation,the dextrin hydrolysis rate will significantly exceed the rate ofglucose uptake and conversion to ethanol by the yeast, resulting insubstantial accumulation of glucose in fermentation (concentrations ofglucose greater than 10%-11% w/v). High concentrations of glucose infermentation can slow yeast growth and fermentation rates due toelevated osmotic pressure applied to the yeast cells; this can result inthe inability of the yeast population to convert all of the availableglucose in fermentation to ethanol within the available fermentationbatch duration. One yeast cellular response to elevated osmotic pressureis to produce higher intracellular concentrations of glycerol toincrease the intracellular osmolyte concentration. The diversion ofcarbon from glucose by the yeast to increased glycerol production,rather than ethanol production, can reduce the ethanol yield achieved infermentation. On the other hand, if the GA enzyme is under-dosed infermentation, in early-, mid-, or late-fermentation (depending on howseverely the GA enzyme is under-dosed) the rate of glucose uptake andconversion to ethanol by the yeast will exceed the rate of glucosegeneration from dextrin hydrolysis to such an extent that the glucoseconcentration will be reduced to nearly zero with a significantconcentration of residual starch dextrins remaining to be hydrolyzed andfermented. If the rate of glucose generation at this point isinsufficient to supply the rate of energy generation by fermentationrequired for yeast cell maintenance, then the viable yeast cellpopulation will begin to decrease. Depending on the rate of decrease inyeast cell viability and the fermentation duration remaining untilfermentation drop, this may either result in a fermentation batch with alow glucose concentration at drop 0.1% w/v), reduced ethanol yield, andelevated concentrations of residual dextrins; or, if the viable yeastpopulation decreases to near zero well before fermentation drop, afermentation batch with an elevated glucose concentration at drop (>0.1%w/v), normal or elevated concentrations of residual dextrins, andreduced ethanol yield.

In some embodiments, the recombinant yeast host cells described hereinreduce the need for (e.g., displace) exogenous enzyme dosing insaccharification/fermentation. As such, a reduced dose of exogenousenzyme can be used in combination with the recombinant yeast host cellof the present disclosure in order to complete the fermentation. Thedose used in combination with the recombinant yeast host cell of thepresent disclosure is considered to be “reduced” because it is lowerthan a control dose necessary for a control yeast host cell (lacking theability to hydrolyze starch or dextrin) to complete the fermentation. Asused in the context of the present disclosure, a “dose necessary tocomplete the fermentation” refers to the amount of an exogenous enzymewhich is required by a yeast host cell (recombinant or control yeasthost cell) to convert the available starch or dextrin in the biomassinto glucose, allowing the yeast host cell to convert the glucose into afermentation product (such as ethanol). As used in the context of thepresent disclosure, a “reduced dose” does not include the amount of theexogenous enzyme that may be used during the mash propagation step andonly refers to the amount of the exogenous enzyme that is used duringfermentation. In some specific embodiments, especially when the controlyeast host cell is Ethanol Red, the control dose allows achieving,during a permissive fermentation, a fermentation yield of at least0.415%, w/v per w/w of biomass. In still another specific embodiment,the reduced dose allows achieving, during a permissive fermentation afermentation yield of at least 0.440%, w/v per w/w of biomass. In someembodiments, the reduced dose of the exogenous enzyme is lower by atleast about 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% on a weight basis whencompared to the control dose. In the context of the present disclosure,the term “about”, when used in combination with the % on a weight basis,refers to ±1%. In still another specific embodiment, the exogenousenzyme is lower by at least about 68% on a weight basis when compared tothe control dose. In yet another embodiment, the process does notinclude adding the exogenous enzyme (e.g., enzyme displacement can be100%). In still another embodiment, the fermentation yield (obtainedwith the reduced dose) is equal to or higher than the controlfermentation yield of the control fermentation (completed by a controlyeast cell lacking the ability to hydrolyze starch or dextrin). In stillanother embodiment, the fermentation yield (obtained with the reduceddose) is substantially similar to the control fermentation yield of thecontrol fermentation (completed by a control yeast cell lacking theability to hydrolyze starch or dextrin). In the context of the presentdisclosure, the expression “substantially similar” when used inconnection to “the control fermentation yield” refers to a fermentationyield (obtained with a reduced dose and the recombinant yeast host cell)that is ±5% of the control fermentation yield of the controlfermentation (completed by a control yeast cell lacking the ability tohydrolyze starch or dextrin).

In some embodiments, the recombinant yeast host cells described hereinreduce (e.g., displaces) the need for exogenous enzyme dosing insimultaneous saccharification and fermentation (SSF). In someembodiments, the recombinant yeast host cells described herein alleviatethe need for exogenous enzyme dosing in SSF. In some embodiments, therecombinant yeast host cells described herein is less susceptible totemperature and pH stress in SSF processes. As such, in some embodimentsof the process, the recombinant yeast host cell of the presentdisclosure allows for a complete displacement of the exogenous purifiedenzyme (which can be in some embodiments a glucoamylase) while achievingthe same fermentation yield as a corresponding control yeast strain(e.g., lacking ability to saccharify the biomass without addition of theexogenous enzyme) in the presence of a full dose of the exogenous enzyme(which can be in some embodiments a glucoamylase). The process can, insome embodiments, alleviate the need to supplement the hydrolyzedbiomass with a exogenous enzyme (which can be in some embodiments aglucoamylase) during the fermentation step.

In some embodiments of the present disclosure, the process can occur ata commercial scale. For example, the process can use a stabilized liquidyeast dose of at least 30 kg, 40 kg, 50 kg, 60 kg, 70 kg, 75 kg, 80 kg,90 kg, 100 kg, 110 kg, 120 kg, 130 kg, 140 kg, 150 kg, 160 kg, 170 kg,180 kg, 190 kg, 200 kg, 210 kg or more. In a specific embodiment, theprocess can use a stabilized liquid yeast dose between 30 and 210 kg,for example between 112.4-168.5 kg. In yet another embodiment, theprocess can use a stabilized liquid yeast dose between 112.4 kg and116.4 kg, 112.4 kg and 128.4 kg, 112.4 kg and 132.4 kg, 112.4 kg and148.5 kg or 112.4 kg and 168.5 kg. In still a further embodiment, theprocess can use a stabilized liquid yeast dose between 116.4 kg and128.4 kg, 116.4 kg and 132.4 kg, 116.4 kg and 148.5 kg or 116.4 and168.5 kg. In still a further embodiment, the process can use astabilized liquid yeast dose between 128.4 kg and 132.4 kg, 128.4 kg and148.5 kg or 128.4 kg and 168.5 kg. In still a further embodiment, theprocess can use a stabilized liquid yeast dose between 132.4 kg and148.5 kg or 132.4 kg and 168.5 kg. In still a further embodiment, theprocess can use a stabilized liquid yeast dose between 148.5 kg and168.5 kg. In yet another embodiment, the process can initially includebetween 28% and 37% solids, for example between 30% and 35% solids orbetween 32% and 33% solids. In yet another embodiment, the fermentationduration (at drop) can be at least 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79 80, 81, 82, 83, 84, 85, 86, 87, 88,89 90 hours or more. In yet another embodiment, the fermentationduration can be about 64.3, 65.5, 67, 68.7, 71.4, 74.6, 81.5, 82.3,82.5, 82.8 hours or more. In commercial application, the process isperformed in a fermentor. As it is known in the art, a “fermentor”refers to the apparatus for carrying our fermentation whereas a“fermenter” refers to the actual organism that causes the fermentation.In yet a further embodiment, the working volume of the fermentationreactor/fermentor can be, for example, at least 100 000, 200 000, 300000, 400 000, 500 000, 600 000, 700 000, 800 000, 900 000, 1 000 000gallons or more. In yet a further embodiment, the working volume of thefermentation reactor can be, for example, at least 500 000, 525 000, 550000, 575 000, 600 000, 625 000, 650 000, 675 000, 700 000, 725 000, 750000, 775 000, 800 000, 825 000, 850 000, 875 000, 900 000 gallons ormore. In yet a further embodiment, the working volume of thefermentation reactor/fermentor can be, for example, between 700 000 and725 000, 700 000 and 750 000, 700 000 and 775 000, 700 000 and 800 000,700 000 and 825 000, 700 000 and 850 000, 700 000 and 875 000 or 700 000and 900 000 gallons. In yet a further embodiment, the working volume ofthe fermentation reactor/fermentor can be, for example, between 725 000and 750 000, 725 000 and 775 000, 725 000 and 800 000, 725 000 and 825000, 725 000 and 850 000, 725 000 and 875 000 or 725 000 and 900 000gallons. In yet a further embodiment, the working volume of thefermentation reactor/fermentor can be, for example, between 750 000 and775 000, 750 000 and 800 000, 750 000 and 825 000, 750 000 and 850 000,750 000 and 875 000 or 750 000 and 900 000 gallons. In yet a furtherembodiment, the working volume of the fermentation reactor/fermentor canbe, for example, between 775 000 and 800 000, 775 000 and 825 000, 775000 and 850 000, 775 000 and 875 000 or 775 000 and 900 000 gallons. Inyet a further embodiment, the working volume of the fermentationreactor/fermentor can be, for example, between 800 000 and 825 000, 800000 and 850 000, 800 000 and 875 000 or 800 000 and 900 000 gallons. Inyet a further embodiment, the working volume of the fermentationreactor/fermentor can be, for example, between 825 000 and 850 000, 825000 and 875 000 or 825 000 and 900 000 gallons. In yet a furtherembodiment, the working volume of the fermentation reactor/fermentor canbe, for example, between 850 000 and 875 000 or 850 000 and 900 000gallons. In yet a further embodiment, the working volume of thefermentation reactor/fermentor can be, for example, between 875 000 and900 000 gallons. In still a further example, especially when thefermentation is a batch fermentation, the fermentor fill duration can beat least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 hours or more. In some embodiments, the fermentor fill duration canbe between 5 and 20 hours.

Yeast Products and Compositions

The recombinant yeast host cells of the present disclosure can be usedin the preparation of a yeast composition (e.g., a compositioncomprising the recombinant yeast host cell) comprising the heterologouspolypeptide having starch or dextrin hydrolase activity. The yeastcompositions and products can be provided in a liquid, semi-liquid ordry form.

A yeast composition refers to a composition comprising the recombinantyeast host cell of the present disclosure (which may be, in someembodiments, a viable recombinant yeast host cell) as well as theheterologous polypeptide having starch or dextrin hydrolase acitivity.The process for providing a yeast composition comprises providing apropagated the recombinant yeast host cell and removing, at least onecomponent of the mixture obtained after propagation to provide the yeastcomposition. This component can be, without limitation, water, aminoacids, peptides and proteins, nucleic acid residues and nucleic acidmolecules, cellular debris, fermentation products, etc. In anembodiment, the process comprises substantially isolating the propagatedrecombinant yeast host cells from the components of the propagationmedium. As used in the context of the present disclosure, the expression“substantially isolating” refers to the removal of the majority of thecomponents of the propagation medium from the propagated recombinantyeast host cells. In some embodiments, “substantially isolating” refersto concentrating the propagated recombinant yeast host cell to at least5, 10, 15, 20, 25, 30, 35, 45% or more when compared to theconcentration of the recombinant yeast host cell prior to the isolation.In order to provide the yeast composition, the propagated recombinantyeast host cells can be centrifuged (and the resulting cellular pelletcomprising the propagated recombinant yeast host cells can optionally bewashed), filtered and/or dried (optionally using a vacuum-dryingtechnique). The isolated recombinant yeast host cells can then beformulated in a yeast composition. The yeast composition can be providedin an active or a semi-active form. The yeast composition can beprovided in a liquid, semi-solid or dry form. In an embodiment, theyeast composition can be provided in the form of a cream yeast. In someembodiments, the process also include propagating the recombinant yeasthost cell prior to the removal step. The yeast composition can beoptionally stored prior to the fermentation phase. In such embodiment,the yeast composition can include, for example, one or more stabilizersor preservatives and, in some embodiment, an unfermentable carbon source(such as trehalose for example).

In some embodiments, the recombinant yeast host cell or the yeastcomposition obtained therefrom can be provided in a composition incombination with starch. Such composition can include additionalexogenous enzyme(s) which may be used during the saccharification and/orfermentation steps.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example I—Heterologous Starch Digesting Glucoamylase in RecombinantYeast Cells

TABLE 1 Genotypes of the strains used in the examples. All therecombinant strains were derived from M2390 and expressed therecombinant enzyme under transcriptional control of the constitutivetef2p promoter and idp1t terminator. Strain name Expressed enzyme Enzymedescription M2390 None - wild-type Saccharomyces cerevisiae strainM17199 MP1152 Glucoamylase from (SEQ ID NO: 9) Saccharomycopsisfibuligera associated with the signal sequence of Saccharomycescerevisiae alpha-mating factor 1 M15621 SEQ ID NO: 6 Glucoamylase fromRasamsonia emersonii associated with its native signal sequence M23176and MP1262 Glucoamylase from Rasamsonia M23177* (SEQ ID NO: 1) emersoniiassociated with the signal sequence of Saccharomyces cerevisiaealpha-mating factor 1 *M23176 and M23177 are two different isolates fromthe same transformation

Permissive corn mash fermentation data. Permissive fermentationconditions were conducted as follows: 32.4% total solids, 300 ppm urea,33° C. (1-48 hours), exogenous glucoamylase GA enzyme inclusion aslisted under each bar of the figures. FIG. 1 illustrates the surprisingresult of MP1262 secreting strains (M23176 and M23177) showingperformance parity at 0% exogenous glucoamylase inclusion to M2390 dosedwith a full 100% dose of exogenous enzyme. Without wishing to be boundto theory, the fact that the ethanol titers show parity for isolatesM23176/M23177 with M2390 suggests that the expression of theheterologous MP1262 does not seem to reduce strain fermentativeperformance.

FIG. 2 further illustrates that the signal peptide optimized MP1262secreting strains can successfully finish fermentation with 100%exogenous enzyme displacement, achieving the same titers as M2390 dosedwith a full 100% GA dose.

In FIG. 3, four strains were compared side by side at either 33% or 0%exogenous enzyme inclusion. The reduction in fermentation performancegoing from 33% to 0% enzyme inclusion for M17199 and M15621 can beattributed to insufficient heterologous GA secretion in the conditionstested. Strain M23177 did not exhibit a reduction in in fermentationperformance in the absence of the exogenous enzyme.

Non-permissive corn mash fermentation data. The fermentations wereconducted according to the following conditions: 32.4% total solids, nourea, 34° C. (1-48 hours) or 36° C. (1-48 hours, for heat-treatmentchallenge only), 0.38% w/v lactic acid added 20 hours into thefermentation (for lactic acid challenge only), exogenous glucoamylase(GA) enzyme inclusion as listed under each bar of the figures.

As shown in FIGS. 4 and 5, strain M23177 did not exhibit a reduction inin fermentation performance in the absence of the exogenous enzymeduring non-permissive fermentation (FIG. 4 shows the results associatedwith a lactic acid challenge and FIG. 5 shows the results associatedwith a heat treatment).

Example II—Enzyme Displacement

The fermentation performances of two recombinant Saccharomycescerevisiae host cell types have been compared to a non-engineered yeaststrain at Production Facility A. Yeast strain M23541 expresses asecreted form of a Rasamsonia emersonii glucoamylase (having the aminoacid sequence of SEQ ID NO: 1). Yeast strain M15419 (corresponding tothe commercial product TransFerm®RB3) expressed a secreted form of aSaccharomycopsis fibuligera (glucoamylase having the amino acid sequenceof SEQ ID NO: 37). Yeast strains M23541 and M15419 both bear additional“glycerol reduction background modifications” described in WO2011140386,WO2012138942 and WO2020100069 (all incorporated herewith in theirentirety) and allowing the reduction of production of glycerol. Table 2summarizes the progression of the testing, and the conditions presentduring each testing phase, from use of conventional yeast. Table 3summarizes the average (mean) measurements for all fermentation batcheswithin trial phases, including average slurry total solids used toproduce liquefact filling fermentation batches, average componentconcentrations (DP4+, DP3, DP2, glucose, glycerol, ethanol, totalresidual sugars) in fermentation beer soluble fraction (supernatant)over the time course of fermentation batches, average ethanol yield perslurry total solids over the time course of fermentation batches(ethanol concentration in fermentation beer soluble fraction normalizedto batch slurry total solids), and average fermentation duration.

TABLE 2 Conditions during progression of testing at Production FacilityA, including: average slurry total solids, average fermentationduration, yeast type added in mash propagation, yeast dose used in mashpropagation, exogenous GA cocktail dose used in mash propagation,exogenous GA cocktail dose used in fermentation, displacement ofexogenous GA cocktail dose in fermentation relative to dose used withconventional yeast (yeast not expressing starch- or dextrin-degradingenzymes), exogenous GA cocktail type used in mash propagation andfermentation, and exogenous alpha-amylase (AA) type used in mashingprocess. Average Average Fermentation Slurry Duration Total Solids forPhase Number of for Phase (at Drop, Week Phase Batches Phase Detail (%w/w) hours) Yeast Type Week 1 A 16 Conventional Yeast, 32% 32.01 67.0Ethanol Red; w/w Slurry Solids Target, Conventional 100 gal Exogenous GAActive Dry Cocktail in Fermentation, Yeast Type B 0.75 gal Exogenous GACocktail in Propagation, 40 kg Active Dry Yeast Dose Week 1-2 B 11M15419, 32% w/w Slurry 31.86 65.5 M15419 Solids Target, 51 gal ExogenousGA Cocktail in Fermentation, 0.75 gal Exogenous GA Cocktail inPropagation, 112.4 kg Stabilized Liquid Yeast Dose Week 2 C 15 M15419,33% w/w Slurry 32.95 64.3 M15419 Solids Target, 51 gal Exogenous GACocktail in Fermentation, 0.75 gal Exogenous GA Cocktail in Propagation,112.4 kg Stabilized Liquid Yeast Dose Week 30 D 3 M23541, 33% w/w Slurry32.99 82.8 M23541 Solids Target, 17 gal Exogenous GA Cocktail inFermentation, 0.5 gal Exogenous GA Cocktail in Propagation, 168.5 kgStabilized Liquid Yeast Dose Week 30 E 1 M23541, 33% w/w Slurry 32.7782.3 M23541 Solids Target, 10 gal Exogenous GA Cocktail in Fermentation,0.5 gal Exogenous GA Cocktail in Propagation, 168.5 kg Stabilized LiquidYeast Dose Weeks 30-31 F 11 M23541, 33% w/w Slurry 32.97 83.3 M23541Solids Target, 10 gal Exogenous GA Cocktail in Fermentation, NoExogenous GA Cocktail in Propagation, 168.5 kg Stabilized Liquid YeastDose Week 31 G 3 M23541, 33% w/w Slurry 33.01 82.5 M23541 Solids Target,10 gal Exogenous GA Cocktail in Fermentation, No Exogenous GA Cocktailin Propagation, 148.5 kg Stabilized Liquid Yeast Dose Week 31 H 1M23541, 33% w/w Slurry 33.03 81.5 M23541 Solids Target, No Exogenous GACocktail in Fermentation, No Exogenous GA Cocktail in Propagation, 148.5kg Stabilized Liquid Yeast Dose Weeks 31-32 I 17 M23541, 33% w/w Slurry33.00 74.6 M23541 Solids Target, 10 gal Exogenous GA Cocktail inFermentation, No Exogenous GA Cocktail in Propagation, 148.5 kgStabilized Liquid Yeast Dose, Shorter Fermentation Durations Weeks 32-33J 28 M23541, 33% w/w Slurry 32.99 71.4 M23541 Solids Target, 10 galExogenous GA Cocktail in Fermentation, No Exogenous GA Cocktail inPropagation, 128.4 kg Stabilized Liquid Yeast Dose, Shorter FermentationDurations Week 33 K 5 M23541, 33% w/w Slurry 33.03 68.7 M23541 SolidsTarget, 10 gal Exogenous GA Cocktail in Fermentation, No Exogenous GACocktail in Propagation, 116.4 kg Stabilized Liquid Yeast Dose, ShorterFermentation Durations Yeast Dose in Mash Propagation DisplacementExogenous (ADY = Total Mash Total of Exogenous Exogenous Alpha-AmylaseActive Dry Propagation Fermentation GA Cocktail GA Cocktail (AA)Cocktail Yeast, SLY = Exogenous Exogenous Dose in Type in Type Used inStabilized GA Cocktail GA Cocktail Fermentation Propagation & MashingWeek Liquid Yeast) Dose (gal) Dose (gal) (%) Fermentation Process Week 140 kg ADY 0.75 100 0.0% Third-Party Third-Party Exogenous Exogenous AAGA Cocktail Cocktail Type A Type A Week 1-2 112.4 kg 0.75 51 49.0%Third-Party Third-Party SLY Exogenous Exogenous AA GA Cocktail CocktailType A Type A Week 2 112.4 kg 0.75 51 49.0% Third-Party Third-Party SLYExogenous Exogenous AA GA Cocktail Cocktail Type A Type A Week 30 168.5kg 0.50 17 83.0% Third-Party Third-Party SLY Exogenous Exogenous AA GACocktail Cocktail Type B Type B Week 30 168.5 kg 0.50 10 90.0%Third-Party Third-Party SLY Exogenous Exogenous AA GA Cocktail CocktailType B Type B Weeks 30-31 168.5 kg 0.00 10 90.0% Third-Party Third-PartySLY Exogenous Exogenous AA GA Cocktail Cocktail Type B Type B Week 31148.5 kg 0.00 10 90.0% Third-Party Third-Party SLY Exogenous ExogenousAA GA Cocktail Cocktail Type B Type B Week 31 148.5 kg 0.00 0 100.0%Third-Party Third-Party SLY Exogenous Exogenous AA GA Cocktail CocktailType B Type B Weeks 31-32 148.5 kg 0.00 10 90.0% Third-Party Third-PartySLY Exogenous Exogenous AA GA Cocktail Cocktail Type B Type B Weeks32-33 128.4 kg 0.00 10 90.0% Third-Party Third-Party SLY ExogenousExogenous AA GA Cocktail Cocktail Type B Type B Week 33 116.4 kg 0.00 1090.0% Third-Party Third-Party SLY Exogenous Exogenous AA GA CocktailCocktail Type B Type B

TABLE 3 Average (mean) measurements for all fermentation batches withintrial phases at Production Facility A, including average slurry totalsolids used to produce liquefact filling fermentation batches, averagecomponent concentrations (DP4+, DP3, DP2, glucose, glycerol, ethanol,total residual sugars) in fermentation beer soluble fraction(supernatant) over the time course of fermentation batches, averageethanol yield per slurry total solids over the time course offermentation batches (ethanol concentration in fermentation beer solublefraction normalized to batch slurry total solids), and averagefermentation duration. Phase A B C D E F G H I J K Displacement ofExogenous GA 0.0% 49.0% 49.0% 83.0% 90.0% 90.0% 90.0% 100.0% 90.0% 90.0%90.0% Enzyme Cocktail Dose in Fermentation (%) Measurement FermentationMean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Sample Time Point(h) Slurry Total Average During 32.01 31.86 32.95 32.99 32.77 32.9733.01 33.03 33.00 32.99 33.03 Solids (% w/w) Fermentor Fill Ethanol (%w/v) 10 h 2.287 2.118 2.017 2.408 2.247 2.552 2.242 2.242 2.360 2.3272.071 18 h 6.166 5.889 5.741 6.360 6.295 6.820 6.541 6.210 6.500 6.3285.750 25 h 8.746 8.398 8.382 9.021 8.924 9.579 9.396 9.095 9.223 8.9868.537 39 h 12.422 11.899 11.840 12.369 12.402 13.261 13.037 12.53612.676 12.522 12.036 52 h 13.214 13.551 13.770 14.456 13.741 14.43614.457 14.065 14.418 14.410 14.140 60 h 13.329 13.789 14.285 14.76914.468 14.452 14.465 14.495 14.583 14.607 14.731 Fermentation 13.35813.790 14.306 14.914 14.656 14.500 14.537 14.544 14.643 14.693 14.812Drop Ethanol Yield 10 h 0.0714 0.0667 0.0612 0.0730 0.0686 0.0774 0.06790.0679 0.0715 0.0706 0.0627 (Batch Ethanol 18 h 0.1926 0.1850 0.17420.1928 0.1921 0.2069 0.1982 0.1880 0.1970 0.1918 0.1741 Concentration 25h 0.2732 0.2637 0.2544 0.2734 0.2723 0.2905 0.2846 0.2753 0.2794 0.27240.2585 Normalized to 39 h 0.3880 0.3737 0.3593 0.3749 0.3785 0.40220.3949 0.3795 0.3841 0.3796 0.3644 Batch Slurry 52 h 0.4128 0.42560.4179 0.4382 0.4193 0.4378 0.4380 0.4258 0.4368 0.4368 0.4281 TotalSolids, 60 h 0.4164 0.4331 0.4335 0.4476 0.4415 0.4383 0.4382 0.43880.4419 0.4428 0.4460 % w/v Ethanol Fermentation 0.4173 0.4331 0.43410.4520 0.4473 0.4398 0.4404 0.4403 0.4437 0.4454 0.4485 at Time PointDrop per % w/w Slurry Total Solids) Fermentation Start of 67.0 65.5 64.382.8 82.3 83.3 82.5 81.5 74.6 71.4 68.7 Duration (h) Fermentor Fill toFermentation Drop

Whereas the conventional yeast not engineered to secrete glucoamylaserequired a 100 gal dose of an exogenous GA enzyme cocktail infermentation to facilitate starch dextrin hydrolysis to glucose andfermentation to ethanol, and yeast strain M15419 required a 51 gal doseof the exogenous GA enzyme cocktail in fermentation (a 49.0%displacement of exogenous GA cocktail dosage relative to conventionalyeast), strain M23541 required between zero exogenous GA in mashpropagation and fermentation to 17 gal of exogenous GA cocktail infermentation (between complete, 100.0% displacement of the exogenous GAcocktail in mash propagation and fermentation, and 83.0% displacement ofthe exogenous GA cocktail in fermentation) (Table 2). As the total finalfermentation durations varied between yeast strain types and conditions,ethanol yield data is presented here for all conditions after both 60 hof fermentation (FIG. 6), and at the end of fermentation (fermentationdrop, FIG. 7).

At 60 h fermentation duration (FIG. 6), use of strain M23541 allowed forstatistically significantly higher ethanol yields (95% confidence level)relative to conventional yeast used at 100 gal of exogenous GA cocktailat all M23541 yeast doses and exogenous GA dose conditions shown(between 116.4 kg and 168.5 kg of M23541 stabilized liquid yeast used toinoculate mash propagation, and between zero exogenous GA enzymecocktail used in mash propagation and fermentation and 17 gal ofexogenous GA cocktail used in fermentation with 0.5 gal of exogenous GAenzyme cocktail used in mash propagation). At 60 h fermentation duration(FIG. 6), use of strain M23541 allowed for statistically equivalent orhigher ethanol yields (95% confidence level) relative to strain M15419used at 51 gal of exogenous GA enzyme cocktail at all M23541 yeast dosesand exogenous GA cocktail dose conditions shown. Use of M23541 at dosesof 10 gal exogenous GA enzyme cocktail in fermentation and at zerogallons of exogenous GA enzyme cocktailin fermentation did not result instatistically significantly different ethanol yields from M23541 used ata 17 gal exogenous GA enzyme cocktail dosed in fermentation.

By the end of fermentation (fermentation drop, FIG. 7), use of strainM23541 allowed for statistically significantly higher ethanol yields(95% confidence level) relative to conventional yeast used at 100 gal ofexogenous GA cocktail at all M23541 yeast doses and exogenous GA doseconditions shown (between 116.4 kg and 168.5 kg of M23541 stabilizedliquid yeast used to inoculate mash propagation, and between zerogallons of exogenous GA enzyme cocktail used in mash propagation andfermentation and 17 gal of exogenous GA enzyme cocktail used infermentation with 0.5 gal of exogenous GA enzyme cocktail used in mashpropagation). By the end of fermentation (FIG. 7), use of strain M23541allowed for statistically equivalent or higher ethanol yields (95%confidence level) relative to strain M15419 used at 51 gal of exogenousGA cocktail at all M23541 yeast doses and exogenous GA enzyme cocktaildose conditions shown. Use of M23541 at doses of 10 gal exogenous GAenzyme cocktail in fermentation and at zero gallons of exogenous GAenzyme cocktail in fermentation did not result in statisticallysignificantly different ethanol yields from M23541 used at a 17 galexogenous GA enzyme cocktail dosed in fermentation. The evolution ofethanol yield by strain and condition tested over the course offermentation duration is summarized in FIG. 8.

By 60 h in fermentation, the extent of hydrolysis and conversion ofsoluble dextrins with use of strain M23541 at all yeast doses andexogenous GA dose conditions shown (between 116.4 kg and 168.5 kg ofM23541 stabilized liquid yeast used to inoculate mash propagation, andbetween zero gallons of exogenous GA enzyme cocktail used in mashpropagation and fermentation and 17 gal of exogenous GA enzyme cocktailused in fermentation with 0.5 gal of exogenous GA enzyme cocktail usedin mash propagation) was equivalent to or better than (lower than) withuse of both conventional yeast (Ethanol Red and a yeast product of anequivalent nature not engineered to express glucoamylase or otherstarch- or dextrin-degrading enzymes) at 100 gal of exogenous GAcocktail and use of strain M15419 at 51 gal of exogenous GA cocktail, asindicated by total residual sugar concentrations at this time.

Example III—Corn Fermentation Using Dry Yeast Samples

Permissive corn mash fermentation. Permissive fermentation conditionswere conducted as follows: 31.55% total solids, 612 ppm urea, 32° C.(0-52 hours), exogenous glucoamylase GA enzyme inclusion as listed undereach bar of the figures. The “100%” enzyme dose is equivalent to 0.6AGU/gTS. Yeast dosing was carried out through direct pitch (0.05 g drycell weight/L inoculum) from dry yeast samples rehydrated in sterilewater at ambient temperature for 30 minutes. The fermentations werecarried out using 10 mL scintillation vials with a total sample size of3 grams.

The fermentation performance of three distinct yeast strains has beendetermined. Once reconstituted, the dry yeasts samples comprisingstrains M23177 (described in Example I), M24926 (expressing the R.emersonii glucoamylase with the alpha-mating factor signal sequence likestrain M23177 and bearing an additional “trehalose reduction” geneticmodification described in U.S. Pat. No. 10,570,421 and incorporatedherewith in their entirety) and M23541 (expressing the R. emersoniiglucoamylase with the alpha-mating factor signal sequence like strainM23177 and bearing additional “glycerol reduction backgroundmodifications” refers the genetic modifications described inWO2011140386, WO2012138942 and WO2020100069 allowing the reduction ofproduction of glycerol, all incorporated herewith in their entirety)were shown to produce ethanol even when a lower dose of exogenousglucoamylase was used (FIG. 9). In FIG. 10, the results obtained in FIG.9 (at 52 hours) were plotted as relative ethanol yield compared to theresult obtained with the conventional strain Ethanol Red (provided in anactive dried form or ADY).

While the invention has been described in connection with specificembodiments thereof, it will be understood that the scope of the claimsshould not be limited by the preferred embodiments set forth in theexamples, but should be given the broadest interpretation consistentwith the description as a whole.

What is claimed is:
 1. A process for fermenting a biomass into afermentation product, the process comprises contacting the biomass witha recombinant yeast host cell and a reduced dose of an exogenous enzyme,under a condition that allows the conversion of at least a part of thebiomass into the fermentation product, wherein: the recombinant yeasthost cell has a heterologous nucleic acid molecule encoding aheterologous polypeptide having starch or dextrin hydrolase activity,wherein the heterologous nucleic acid molecule comprises a firstpolynucleotide encoding a heterologous signal sequence; and a secondpolynucleotide encoding a heterologous polypeptide having starch ordextrin hydrolase activity; the first polynucleotide molecule isoperatively associated with the second polynucleotide molecule; theexogenous enzyme is a starch or dextrin hydrolase; and the reduced doseof the exogenous enzyme is lower than a control dose necessary for acontrol yeast host cell lacking the ability to hydrolyze starch ordextrin to complete a corresponding control fermentation.
 2. The processof claim 1, wherein the control yeast host cell lacks the heterologousnucleic acid molecule.
 3. The process of claim 1, wherein theheterologous polypeptide having starch or dextrin hydrolase activity isan heterologous polypeptide having glucoamylase activity.
 4. The processof claim 3, wherein the polypeptide having glucoamylase activity has theamino acid sequence of SEQ ID NO: 3 or 13, is a variant of the aminoacid sequence of SEQ ID NO: 3, 13 having glucoamylase activity, or is afragment of the amino acid sequence of SEQ ID NO: 3 or 13 havingglucoamylase activity.
 5. The process of claim 1, wherein theheterologous signal sequence has the amino acid sequence of SEQ ID NO:5, is a variant of the amino acid sequence of SEQ ID NO: 5 having signalsequence activity, or is a fragment of the amino acid sequence of SEQ IDNO: 5 having signal sequence activity.
 6. The process of claim 1,wherein the heterologous nucleic acid molecule encodes the heterologouspolypeptide having the amino acid sequence of SEQ ID NO: 1 or 11, avariant of the amino acid sequence of SEQ ID NO: 1 or 11 havingglucoamylase activity, or a fragment of the amino acid sequence of SEQID NO: 1 or 11 having glucoamylase activity.
 7. The process of claim 1,wherein the heterologous nucleic acid molecule further comprises a thirdpolynucleotide comprising a heterologous promoter operatively associatedwith the first polynucleotide and the second polynucleotide allowing theexpression of the heterologous polypeptide having starch or dextrinhydrolase activity.
 8. The process of claim 7, wherein the heterologouspromoter is capable of allowing the expression of the heterologouspolypeptide having starch or dextrin hydrolase activity duringpropagation.
 9. The process of claim 8, wherein the heterologouspolypeptide having starch or dextrin activity is a secreted polypeptide.10. The process of claim 8, wherein the heterologous polypeptide havingstarch or dextrin activity is a membrane-associated polypeptide.
 11. Theprocess of claim 10, wherein the membrane-associated polypeptide is atethered polypeptide.
 12. The process of claim 1, wherein therecombinant yeast host cell comprises a further heterologous nucleicacid molecule encoding a heterologous alpha-amylase and/or aheterologous glucoamylase.
 13. The process of claim 12, wherein theheterologous alpha-amylase has the amino acid sequence of any one of SEQID NO: 17 to 26, is a variant of the amino acid sequence of any one ofSEQ ID NO: 17 to 26 having alpha-amylase activity or is a fragment ofthe amino acid sequence of any one of SEQ ID NO: 17 to 26 havingalpha-amylase activity.
 14. The process of claim 12, wherein theheterologous glucoamylase has the amino acid sequence of any one of SEQID NO: 27 to 36, a variant of the amino acid sequence of any one of SEQID NO: 27 to 36 having glucoamylase activity or a fragment of the aminoacid sequence of any one of SEQ ID NO: 27 to 36 having glucoamylaseactivity.
 15. The process of claim 1, wherein the recombinant yeast hostcell is from the genus Saccharomyces.
 16. The process of claim 15,wherein the recombinant yeast host cell is from the speciesSaccharomyces cerevisiae.
 17. The process of claim 1, wherein thebiomass comprises starch or a starch derivative.
 18. The process ofclaim 17, wherein the biomass is derived from or comprises corn, potato,cassava, rice, wheat, lignocellulosic material, milo or buckwheat. 19.The process of claim 18, wherein the biomass is derived from orcomprises corn.
 20. The process of claim 19, wherein the biomasscomprises or is corn mash.
 21. The process of claim 1, wherein thefermentation product is ethanol.
 22. The process of claim 1, wherein thecontrol dose allows achieving a fermentation yield of at least 0.415%,w/v per w/w of biomass.
 23. The process of claim 1, wherein the reduceddose allows achieving a fermentation yield of at least 0.440%, w/v perw/w of biomass.
 24. The process of claim 1, wherein the reduced dose ofthe exogenous enzyme is lower by at least 50%, 55%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% when compared to the control dose.
 25. The process of claim 1excluding including the exogenous enzyme in the biomass prior to orduring fermentation.
 26. The process of claim 1, wherein the reduceddose of the exogenous enzymes is reduced to zero.
 27. The process ofclaim 1, wherein the fermentation yield is equal to or higher than thecontrol fermentation yield of the control fermentation.
 28. The processof claim 1, wherein the fermentation yield is substantially similar tothe control fermentation yield of the control fermentation.
 29. Theprocess of claim 1, wherein the exogenous enzyme is a glucoamylase. 30.The process of claim 1 occurring at a commercial scale.